Conformationally constrained backbone cyclized peptide analogs

ABSTRACT

Novel backbone cyclized peptide analogs are formed by means of bridging groups attached via the alpha nitrogens of amino acid derivatives to provide novel non-peptidic linkages. Novel building units disclosed are N α (ω-functionalized) amino acids constructed to include a spacer and a terminal functional group. One or more of these N α (ω-functionalized) amino acids are incorporated into a peptide sequence, preferably during solid phase peptide synthesis. The reactive terminal functional groups are protected by specific protecting groups that can be selectively removed to effect either backbone-to-backbone or backbone-to-side chain cyclizations. The invention is specifically exemplified by backbone cyclized bradykinin antagonists having biological activity. Further embodiments of the invention are somatostatin analogs having one or two ring structures involving backbone cyclization.

FIELD OF THE INVENTION

[0001] The present invention relates to conformationally constrainedN^(α) backbone-cyclized peptide analogs cyclized via novel non-peptidiclinkages, to novel N^(α), ω-functionalized amino acid building units, toprocesses for the preparation of these backbone cyclized peptides andbuilding units, to methods for using these peptide analogs and topharmaceutical compositions containing same.

BACKGROUND OF THE INVENTION

[0002] Peptidomimetics

[0003] As a result of major advances in organic chemistry and inmolecular biology, many bioactive peptides can now be prepared inquantities sufficient for pharmacological and clinical utilities. Thusin the last few years new methods have been established for thetreatment and therapy of illnesses in which peptides have beenimplicated. However, the use of peptides as drugs is limited by thefollowing factors: a) their low metabolic stability towards proteolysisin the gastrointestinal tract and in serum; b) their poor absorptionafter oral ingestion, in particular due to their relatively highmolecular mass or the lack of specific transport systems or both; c)their rapid excretion through the liver and kidneys; and d) theirundesired side effects in non-target organ systems, since peptidereceptors can be widely distributed in an organism.

[0004] Moreover, with few exceptions, native peptides of small to mediumsize (less than 30-50 amino acids) exist unordered in dilute aqueoussolution in a multitude of conformations in dynamic equilibrium whichmay lead to lack of receptor selectivity, metabolic susceptibilities andhamper attempts to determine the biologically active conformation. If apeptide has the biologically active conformation per se, i.e.,receptor-bound conformation, then an increased affinity toward thereceptor is expected, since the decrease in entropy on binding is lessthan that on the binding of a flexible peptide. It is thereforeimportant to strive for and develop ordered, uniform and biologicallyactive peptides.

[0005] In recent years, intensive efforts have been made to developpeptidomimetics or peptide analogs that display more favorablepharmacological properties than their prototype native peptides. Thenative peptide itself, the pharmacological properties of which have beenoptimized, generally serves as a lead for the development of thesepeptidomimetics. However, a major problem in the development of suchagents is the discovery of the active region of a biologically activepeptide. For instance, frequently only a small number of amino acids(usually four to eight) are responsible for the recognition of a peptideligand by a receptor. Once this biologically active site is determined alead structure for development of peptidomimetic can be optimized, forexample by molecular modeling programs.

[0006] As used herein, a “peptidomimetic” is a compound that, as aligand of a receptor, can imitate (agonist) or block (antagonist) thebiological effect of a peptide at the receptor level. The followingfactors should be considered to achieve the best possible agonistpeptidomimetic a) metabolic stability, b) good bioavailability, c) highreceptor affinity and receptor selectivity, and d) minimal side effects.

[0007] From the pharmacological and medical viewpoint it is frequentlydesirable to not only imitate the effect of the peptide at the receptorlevel (agonism) but also to block the receptor when required(antagonism). The same pharmacological considerations for designing anagonist peptidomimetic mentioned above hold for designing peptideantagonists, but, in addition, their development in the absence of leadstructures is more difficult. Even today it is not unequivocally clearwhich factors are decisive for the agonistic effect and which are forthe antagonistic effect.

[0008] A generally applicable and successful method recently has beenthe development of conformationally restricted peptidomimetics thatimitate the receptor-bound conformation of the endogenous peptideligands as closely as possible (Rizo and Gierasch, Ann. Rev. Biochem.,61:387, 1992). Investigations of these types of analogs show them tohave increased resistance toward proteases, that is, an increase inmetabolic stability, as well as increased selectivity and thereby fewerside effects (Veber and Friedinger, Trends Neurosci., p. 392, 1985).

[0009] Once these peptidomimetic compounds with rigid conformations areproduced, the most active structures are selected by studying theconformation-activity relationships. Such conformational constraints caninvolve short range (local) modifications of structure or long range(global) conformational restraints (for review see Giannis and Kolter,Angew. Chem. Int. Ed. Enql. 32:1244, 1993).

[0010] Conformationally Constrained Peptides

[0011] Bridging between two neighboring amino acids in a peptide leadsto a local conformational modification, the flexibility of which islimited in comparison with that of regular dipeptides. Somepossibilities for forming such bridges include incorporation of lactamsand piperazinones. γ-Lactams and δ-lactams have been designed to someextent as “turn mimetics”; in several cases the incorporation of suchstructures into peptides leads to biologically active compounds.

[0012] Global restrictions in the conformation of a peptide are possibleby limiting the flexibility of the peptide strand through cyclization(Hruby et al., Biochem. J., 268:249, 1990). Not only does cyclization ofbioactive peptides improve their metabolic stability and receptorselectivity, cyclization also imposes constraints that enhanceconformational homogeneity and facilitates conformational analysis. Thecommon modes of cyclization are the same found in naturally occurringcyclic peptides. These include side chain to side chain cyclization orside chain to end-group cyclization. For this purpose, amino acid sidechains that are not involved in receptor recognition are connectedtogether or to the peptide backbone. Another common cyclization is theend-to-end cyclization.

[0013] Three representative examples are compounds wherein partialstructures of each peptide are made into rings by linking twopenicillamine residues with a disulfide bridge (Mosberg et al., P.N.A.S.US, 80:5871, 1983), by formation of an amide bond between a lysine andan aspartate group (Charpentier et al., J. Med. Chem. 32:1184, 1989), orby connecting two lysine groups with a succinate unit (Rodriguez et al.,Int. J. Pept. Protein Res. 35:441, 1990). These structures have beendisclosed in the literature in the case of a cyclic enkephalin analogwith selectivity for the δ-opiate receptor (Mosberg et al., ibid.); oras agonists to the cholecystokinin B receptor, found largely in thebrain (Charpentier et al., ibid., Rodriguez et al., ibid.).

[0014] The main limitations to these classical modes of cyclization arethat they require substitution of amino acid side chains in order toachieve cyclization.

[0015] Another conceptual approach to the conformational constraint ofpeptides was introduced by Gilon, et al., (Biopolymers, 31:745, 1991)who proposed backbone to backbone cyclization of peptides. Thetheoretical advantages of this strategy include the ability to effectcyclization via the carbons or nitrogens of the peptide backbone withoutinterfering with side chains that may be crucial for interaction withthe specific receptor of a given peptide. While the concept wasenvisaged as being applicable to any linear peptide of interest, inpoint of fact the limiting factor in the proposed scheme was theavailability of suitable building units that must be used to replace theamino acids that are to be linked via bridging groups. The actualreduction to practice of this concept of backbone cyclization wasprevented by the inability to devise any practical method of preparingbuilding units of amino acids other than glycine (Byk et al., J. Org.Chem., 587:5687, 1992). While analogs of other amino acids wereattempted the synthetic method used was unsuccessful or of such lowyield as to preclude any general applicability.

[0016] In Gilon, EPO Application No. 564,739 A2; and J. Org. Chem.,57:5687, 1992, two basic approaches to the synthesis of building unitsare described. The first starts with the reaction of a diamine with ageneral α bromo acid. Selective protection of the ω amine and furtherelaborations of protecting groups provides a building unit, suitable forBoc chemistry peptide synthesis. The second approach starts withselective protection of a diamine and reaction of the product withchloroacetic acid to provide the protected glycine derivative, suitablefor Fmoc peptide synthesis.

[0017] Both examples deal with the reaction of a molecule of the generaltype X—CH(R)—CO—OR′ (wherein X represents a leaving group which, in theexamples given, is either Br or Cl) with an amine which replaces the X.The amine bears an alkylidene chain which is terminated by anotherfunctional group, amine in the examples described, which may or may notbe blocked by a protecting group.

[0018] In all cases the a nitrogen of the end product originates in themolecule which becomes the bridging chain for subsequent cyclization.This approach was chosen in order to take advantage of the highersusceptibility to nucleophilic displacement of a leaving group next to acarboxylic group.

[0019] In a molecule where R is different than hydrogen there is a hightendency to eliminate HX under basic conditions. This side reactionreduces the yield of Gilon's method to the point where it is impracticalfor production of building units based on amino acids other thanglycine. The diamine nitrogen is primary while the product contains asecondary nitrogen, which is a better nucleophile. So while the desiredreaction may be sluggish, and require the addition of catalysts, theproduct may be contaminated with double alkylation products. There is nomention of building units with end group chemistries other thannitrogen, so the only cyclization schemes possible are backbone to sidechain and backbone to C terminus.

[0020] Applications of Conformationally Constrained Peptides

[0021] Conformationally constrained peptides find many pharmacologicaluses. Somatostatin is a cyclic tetradecapeptide found both in thecentral nervous system and in peripheral tissues. It was originallyisolated from mammalian hypothalamus and identified as an importantinhibitor of growth hormone secretion from the anterior pituitary. Itsmultiple biological activities include inhibition of the secretion ofglucagon and insulin from the pancreas, regulation of most gut hormonesand regulation of the release of other neurotransmitters involved inmotor activity and cognitive processes throughout the central nervoussystem (for review see Lamberts, Endocrine Rev., 9:427, 1988).

[0022] Natural somatostatin (also known as Somatotropin ReleaseInhibiting Factor, SRIF) of the following structure:H-Ala¹-Gly²-Cys³-Lys⁴-Asn⁵-Phe⁶-Phe⁷-Trp⁸-Lys⁹-Thr¹⁰-Phe¹¹-Thr¹²-Ser¹³-Cys¹⁴-OH

[0023] was first isolated by Guillemin and colleagues (Brazeau et al.Science, 179:78, 1973). In its natural form, it has limited use as atherapeutic agent since it exhibits two undesirable properties: poorbioavailability and short duration of action. For this reason, greatefforts have been made during the last two decades to find somatostatinanalogs that will have superiority in either potency, biostability,duration of action or selectivity with regard to inhibition of therelease of growth hormone, insulin or glucagon.

[0024] Structure-activity relation studies, spectroscopic techniquessuch as circular dichroism and nuclear magnetic resonance, and molecularmodeling approaches reveal the following: the conformation of the cyclicpart of natural somatostatin is most likely to be an antiparallelβ-sheet; Phe⁶ and Phe¹¹ play an important role in stabilizing thepharmacophore conformation through hydrophobic interactions between thetwo aromatic rings; the four amino acids Phe⁷-Trp⁸-Lys⁹-Thr¹⁰ which arespread around the β-turn in the antiparallel β-sheet are essential forthe pharmacophore; and (D)Trp⁸ is almost always preferable to (L)Trp⁸.

[0025] Nevertheless, a hexapeptide somatostatin analog containing thesefour amino acids anchored by a disulfide bridge:

[0026] is almost inactive both in vitro and in vivo, although it has theadvantage of the covalent disulfide bridge which replaces the Phe⁶-Phe¹¹hydrophobic interactions in natural somatostatin.

[0027] Four main approaches have been attempted in order to increase theactivity of this hexapeptide somatostatin analog. (1) Replacing thedisulfide bridge by a cyclization which encourages a cis-amide bond, orby performing a second cyclization to the molecule yielding a bicyclicanalog. In both cases the resultant analog has a reduced number ofconformational degrees of freedom. (2) Replacing the original aminoacids in the sequence Phe⁷-(D)Trp⁸-Lys⁹-Thr¹⁰ with more potent aminoacid analogs, such as replacing Phe⁷ with Tyr⁷ and Thr¹⁰ with Val¹⁰. (3)Incorporating additional structural elements from natural somatostatinwith the intention that these new elements will contribute to theinteraction with the receptor. (4) Eliminating one of the four aminoacids Phe⁷-(D)Trp⁸-Lys⁹-Thr¹⁰ with the assumption that such analogswould be more selective.

[0028] The somatostatin analog, MK-678:

cyclo(N-Me-Ala⁷-Tyr⁷-(D)Trp⁸-Lys⁹-Val¹⁰-Phe)

[0029] is an example of a highly potent somatostatin analog designedusing the first three approaches above (Lymangrover, et al., LifeScience, 34:371, 1984). In this hexapeptide analog, a cis-amide bond islocated between N-Me-Ala and Phe¹¹, Tyr⁷ and Val¹⁰ replace Phe⁷ andThr¹⁰ respectively, and Phe¹¹ is incorporated from natural somatostatin.

[0030] Another group of somatostatin analogs (U.S. Pat. Nos. 4,310,518and 4,235,886) includes octreotide:

[0031] the only somatostatin analog currently available. It wasdeveloped using the third approach described above. Here, (D)Phe⁵ andthe reduced C-terminal Thr¹²-CH₂OH are assumed to occupy some of theconformational space available to the natural Phe⁶ and Thr¹²,respectively.

[0032] The compound TT2-32:

[0033] is closely related to octreotide and is an example ofimplementing the fourth approach described above. The lack of Thr¹⁰ isprobably responsible for its high selectivity in terms of antitumoractivity.

[0034] These examples of highly potent somatostatin analogs indicatethat the phenylalanines in positions 6 and 11 not only play an importantrole in stabilizing the pharmacophore conformation but also have afunctional role in the interaction with the receptor. It is still anopen question whether one phenylalanine (either Phe⁶ or Phe¹¹) issufficient for the interaction with the receptor or whether both areneeded.

[0035] It is now known that the somatostatin receptors constitute afamily of five different receptor subtypes (Bell and Reisine, TrendsNeurosci., 16, 34-38, 1993), which may be distinguished on the basis oftheir tissue specificity and/or biological activity. Somatostatinanalogs known in the art may not provide sufficient selectivity orreceptor subtype selectivity, particularly as anti-neoplastic agents(Reubi and Laissue, TIPS, 16, 110-115, 1995).

[0036] Symptoms associated with metastatic carcinoid tumors (flushingand diarrhea) and vasoactive intestinal peptide (VIP) secreting adenomas(watery diarrhea) are treated with somatostatin analogs. Somatostatinhas been also approved for the treatment of severe gastrointestinalhemorrhages. Somatostatin may also be useful in the palliative treatmentof other hormone-secreting tumors (e.g., pancreatic islet-cell tumorsand acromegaly) and hormone dependent tumors (e.g., chondrosarcoma andosteosarcoma) due to its anti-secretory activity.

[0037] Another important peptide, Bradykinin, is a naturally occurringnonapeptide, Arg¹-Pro²-Pro³-Gly⁴-Phe⁵-Ser⁶-Pro⁷-Phe⁸-Arg⁹, formed andreleased from precursors in the blood in response to inflammatorystimuli. Elevated levels of bradykinin also appear in other body fluidsand tissues in pathological states such as asthma, septic shock andcommon cold. No clinical abnormalities have been associated so far withbradykinin deficiency which indicates that bradykinin may not play acritical role in normal physiology.

[0038] However, bradykinin mediates its physiological activities bybinding to a specific receptive molecule called the bradykinin receptor.Two such bradykinin receptors have been identified so far (these arecalled B1 and B2 receptors). Subsequent to binding, the bradykininsignal transduction pathway includes production of prostaglandins andleukotrienes as well as calcium activation. Through these mediators,bradykinin is involved in pain, inflammation, allergic reactions andhypotension. Therefore, a substance that can block the ability ofbradykinin to bind to its receptor, namely a bradykinin antagonist,should have a significant therapeutic value for one of the followingindications: asthma, inflammation, septic shock, pain, hypotension andallergy.

[0039] The analog used herein to exemplify backbone cyclization is:

[0040] D-Arg⁰-Arg-R¹-Hyp³-Gly-Phe-R²-D-Phe-Phe⁷-Arg

[0041] (wherein, R¹ is Pro, R² is Ser in native bradykinin). The changeof proline at position 7 of native bradykinin to D-Phe confersantagonist activity. This compound was described in Steranka, et al.,P.N.A.S. U.S., 85:3245-3249, 1988 and is one of a plethora of candidatesequences for modification by the current technology, i.e. backbonecyclization. In this regard, it is worth noting the applications: WO89/01781, EP-A-0370453 and EP-A-0334244 which disclose a widerange ofcandidate structures. Antagonist peptides on which stability and/ortissue selectivity can be conferred by appropriate cyclization will beselected from the many such known sequences.

[0042] According to the present invention a novel synthetic approach isdisclosed providing N^(α)(ω(functionalized)alkylene) amino acid buildingunits that can be used to synthesize novel N^(α)-backbone cyclizedpeptide analogs such as, but not limited to, novel somatostatin andbradykinin analogs. None of the above-mentioned references teaches orsuggests N^(α)-(ω(functionalized)alkylene) amino acids or the novelN^(α)-backbone cyclized peptide analogs of the present invention.

SUMMARY OF THE INVENTION

[0043] It is an object of the present invention to provide backbonecyclized peptide analogs that comprise peptide sequences whichincorporate at least two building units, each of which contains onenitrogen atom of the peptide backbone connected to a bridging group asdescribed below. In the present invention, one or more pairs of thebuilding units is joined together to form a cyclic structure. Thus,according to one aspect of the present invention, backbone cyciizedpeptide analogs are provided that have the general Formula (I):

[0044] wherein: a and b each independently designates an integer from 1to 8 or zero; d, e, and f each independently designates an integer from1 to 10; (AA) designates an amino acid residue wherein the amino acidresidues in each chain may be the same or different; E represents ahydroxyl group, a carboxyl protecting group or an amino group, or CO—Ecan be reduced to CH₂—OH; R, R′, R″, and R′″ each designates an aminoacid side-chain such as H, CH₃, etc., optionally bound with a specificprotecting group; and the lines independently designate a bridging groupof the Formula: (i)-X-M-Y-W-Z-; or (ii)-X-M-Z- wherein: one line may beabsent; M and W are independently selected from the group consisting ofdisulfide, amide, thioether, thioesters, imines, ethers and alkenes; andX, Y and Z are each independently selected from the group consisting ofalkylene, substituted alkylene, arylene, homo- or hetero-cycloalkyleneand substituted cycloalkylene.

[0045] In certain preferred embodiments, the CO—E group of Formula (I)is reduced to a CH₂OH group.

[0046] Another embodiment of the present invention involves N-backboneto side chain cyclized peptides of the general formula (II):

[0047] wherein the substituents are as defined above.

[0048] A preferred embodiment of the present invention involves thebackbone cyclized peptide analog of Formulae I or II wherein the linedesignates a bridging group of the Formula:—(CH₂)_(x)—M—(CH₂)_(y)—W—(CH₂)_(z)— wherein M and W are independentlyselected from the group consisting of disulfide, amide, thioether,thioesters, imines, ethers and alkenes; x and z each independentlydesignates an integer from 1 to 10, and y is zero or an integer of from1 to 8, with the proviso that if y is zero, W is absent.

[0049] Further preferred are backbone cyclized peptide analogs of theFormula I or II wherein R and R′ are other than H. such as CH₃,(CH₃)₂CH—, (CH₃)₂CHCH₂—, CH₃CH₂CH (CH₃)—, CH₃S (CH₂)₂—, HOCH₂—, CH₃CH(OH)—, HSCH₂—, NH₂C(═O) CH₂—, NH₂C(═O)(CH₂)₂—, NH₂(CH₂)₃—, HOC(═O)CH₂—,HOC(═O)(CH₂)₂—, NH₂(CH₂)₄—, C(NH₂)₂NH(CH₂)₃—, HO-phenyl-CH₂—, benzyl,methylindole, and methylimidazole.

[0050] A more preferred embodiment of the present invention is directedto backbone cyclization to stabilize the β-turn conformation ofbradykinin analogs of the general Formula (III):

[0051] wherein M is an amide bond, x and z are each independently aninteger of 1 to 10, and K is H or an acyl group.

[0052] Also more preferred are backbone cyclized peptide analogs of thepresent invention comprising bradykinin analogs of the general Formula(IVa):

[0053] wherein M is an amide bond, x and z are each independently aninteger of 1 to 10, K is H or an acyl group, and R⁶ is Gly or Ser; orthe general Formula (IVb):

[0054] wherein x is an integer of 1 to 10; K is H or an acyl group; (R⁶)is selected from the group of D-Asp, L-Asp, D-Glu and L-Glu; and z isaccording to the amino acid specified: 1 in case of D and L-Asp, and 2in the case of D and L Glu.

[0055] Further more preferred backbone cyclized peptide analogsaccording to the present invention having bradykinin antagonist activityhave the Formula (V):

[0056] wherein M is an amide bond, x and z are each independently aninteger of 1 to 10, and K is H or an acyl group.

[0057] Specifically preferred backbone cyclized peptide analogs of thepresent invention are:

[0058] 1)Ada-(D)Arg-Arg-cyclo(N^(α)(1-(6-aminohexylene)Gly-Hyp-Phe-D-Asp)-D-Phe-Phe-Arg-OH;

[0059] 2)H-D-Arg-Arg-cyclo(N^(α)(1-(4-propanoyl))Gly-Hyp-Phe-N^(α)(3-amido-propylene)Gly)-Ser-D-Phe-Phe-Arg-OH;and

[0060] 3)H-D-Arg-Arg-cyclo(N^(α)(4-propanoyl)Gly-Hyp-Phe-N^(α)(3-amido-propyl)-S-Phe)-Ser-D-Phe-Phe-Arg-OH.

[0061] Another preferred aspect of the present invention is directed tobackbone cyclization to generate novel somatostatin analogs linkedbetween positions 6 and 11, leaving the phenylalanine side chainsuntouched. This conformational stabilization is much more rigid than thePhe⁶, Phe¹¹ hydrophobic interaction in natural somatostatin and is morestable to reduction/oxidation reactions than the Cys-Cys disulfidebridge. In other words, for the first time a stable covalent bridge canbe achieved while either one or both of the original Phe⁶ and Phe¹¹ areretained.

[0062] Moreover, backbone cyclizations can also be used to anchor theβ-turn, not only in positions 6 and 11 but also inside the activereaction of Phe⁷-(D)Trp⁸-Lys⁹-Thr¹⁰, yielding either a monocyclic analogwith a preferable conformation or a very rigid bicyclic analog. Hereagain, the side chains of the pharmacologically active amino acidsremain untouched and the only change is in limiting the conformationalspace.

[0063] As used herein and in the claims in the following more preferredbackbone cyclized peptide analogs, the superscript numbers following theamino acids refer to their position numbers in the native Somatostatin.

[0064] A more preferred backbone cyclized peptide novel analog is theFormula (XIVa):

[0065] with a most preferred analog being the Formula (XIVb):

[0066] wherein m and n are 1, 2 or 3; X is CH₂OH or CONH₂; R⁵ is absentor is Gly, (D)- or (L)-Ala, Phe, Nal and β-Asp(Ind); R⁶ and R¹¹ areindependently Gly or (D)- or (L)-Phe; R⁷ is Phe or Tyr; R¹⁰ is absent oris Gly, Abu, Thr or Val; R¹² is absent or is Thr or Nal, and Y² isselected from the group consisting of amide, disulfide, thioether,imines, ethers and alkenes. In these monocyclic somatostatin analogs, abackbone cyclization replaces the Cys⁶-Cys¹¹ disulfide bridge, leavingthe phenylalanine side chains as in the natural somatostatin. Still morepreferred is the analog wherein Phe⁷ is replaced with Tyr⁷ and Thr¹⁰ isreplaced with Val¹⁰.

[0067] Other more preferred monocyclic analogs that anchor the moleculein positions inside the active region rather than in positions 6 and 11are formulae XV (a and b) and XVI (a-c):

[0068] wherein i and j are independently 1, 2 or 3; X is CH₂OH or CONH₂;R⁵ is absent or is (D)- or (L)-Phe, Nal, or β-Asp(Ind); R⁶ is (D) or(L)-Phe; R¹⁰ is absent or is Gly, Abu or Thr; and R¹¹ is (D)- or(L)-Phe; R¹² is absent or is Thr or Nal, and Y¹ is selected from thegroup consisting of amide, disulfide, thioether, imines, ethers andalkenes.

[0069] Still other more preferred analogs incorporate backbonecyclization in positions 6 and 11 as in Formula XIV, together with thebackbone cyclizations as in Formula XV and XVI, yielding rigid bicyclicanalogs of the Formulae XVII (a and b) and XVIII (a and b):

[0070] wherein i, j, m and n are independently 1, 2 or 3; X is CH₂OH orCONH₂; R⁵ is absent or is (D)- or (L)-Phe, Nal, or β-Asp(Ind); R⁶ andR¹¹ are independently Gly or (D)- or (L)-Phe; R¹⁰ is absent or is Gly,Abu, Val or Thr; R¹² is absent or is Thr or Nal; and Y¹ and Y² areindependently selected from the group consisting of amide, disulfide,thioether, imines, ethers and alkenes.

[0071] Other more preferred bicyclic analogs differ from Formulae XVIIand XVIII by the replacement of the amino acids at positions 6 and 11 bycysteines which form a disulfide bond, leaving only one backbonecyclization in the Formulae XIX (a and b) and XX (a and b):

[0072] wherein i and j are independently 1, 2 or 3; X is CH₂OH or NH₂;R⁵ is absent or is (D)- or (L)-Phe, Nal, or β-Asp(Ind); R⁶ and R¹¹ areindependently Gly or Phe; R¹⁰ is absent or is Gly, Abu or Thr; R¹² isabsent or is Thr or Nal; and Y¹ is selected from the group consisting ofamide, disulfide, thioether, imines, ethers and alkenes.

[0073] Another aspect of the present invention is a method for thepreparation of cyclic peptides of the general Formula (I):

[0074] wherein: a and b each independently designates an integer from 1to 8 or zero; d, e, and f each independently designates an integer from1 to 10; (AA) designates an amino acid residue wherein the amino acidresidues in each chain may be the same or different; E represents ahydroxyl group, a carboxyl protecting group or an amino group, or CO—Ecan be reduced to CH₂—OH; R, R′, R″, and R′″ each designates an aminoacid side-chain optionally bound with a specific protecting group; andthe lines designate a bridging group of the Formula:

(i) —X—M—Y—W—Z—; or (ii) —X—M—Z—

[0075] wherein: one line may be absent; M and W are independentlyselected from the group consisting of disulfide, amide, thioether,thioesters, imines, ethers and alkenes; and X, Y and Z are eachindependently selected from the group consisting of alkylene,substituted alkylene, arylene, homo-or hetero-cycloalkylene andsubstituted cycloalkylene. This method comprises the steps ofincorporating at least one N^(α)-ω-functionalized derivative of aminoacids of Formula (VI):

[0076] wherein X is a spacer group selected from the group consisting ofalkylene, substituted alkylene, arylene, cycloalkylene and substitutedcycloalkylene; R′ is an amino acid side chain, optionally bound with aspecific protecting group; B is a protecting group selected from thegroup consisting of alkyloxy, substituted alkyloxy, or aryl carbonyls;and G is a functional group selected from the group consisting ofamines, thiols, alcohols, carboxylic acids and esters, aldehydes,alcohols and alkyl halides; and A is a specific protecting group of G;into a peptide sequence and subsequently selectively cyclizing thefunctional group with one of the side chains of the amino acids in saidpeptide sequence or with another ω-functionalized amino acid derivative.

[0077] A further object of the present invention is directed to buildingunits known as a N^(α)-ω-functionalized derivatives of the generalFormula (VI) of amino acids which are prerequisites for the cyclizationprocess:

[0078] wherein X is a spacer group selected from the group consisting ofalkylene, substituted alkylene, arylene, cycloalkylene and substitutedcycloalkylene; R is the side chain of an amino acid, optionally boundwith a specific protecting group; B is a protecting group selected fromthe group consisting of alkyloxy, substituted alkyloxy, or aryloxycarbonyls; and G is a functional group selected from the groupconsisting of amines, thiols, alcohols, carboxylic acids and esters,aldehydes and alkyl halides; and A is a protecting group thereof.

[0079] Preferred building units are the ω-functionalized amino acidderivatives wherein X is alkylene; G is a thiol group, an amine group ora carboxyl group; R is phenyl, methyl or isobutyl; with the proviso thatwhen G is an amine group, R is other than H.

[0080] Further preferred are ω-functionalized amino acid derivativeswherein R is protected with a specific protecting group.

[0081] More preferred are ω-functionalized amino acid derivatives of theFormulae:

[0082] wherein X, R, A and B are as defined above.

[0083] Specifically preferred ω-functionalized amino acid derivativesinclude the following:

[0084] 1) N^(α)-(Fmoc)(3-Boc-amino propylene)-(S)Phenylalanine;

[0085] 2) N^(α)-(Fmoc)(3-Boc-amino propylene)-(R)Phenylalanine;

[0086] 3) N^(α)-(Fmoc)(4-Boc-amino butylene)-(S)Phenylalanine;

[0087] 4) N^(α)-(Fmoc)(3-Boc-amino propylene)-(S)Alanine;

[0088] 5) N^(α)-(Fmoc)(6-Boc-amino hexylene)-(S)Alanine;

[0089] 6) N^(α)-(Fmoc)(3-Boc-amino propylene)-(R)Alanine;

[0090] 7) N^(α)-(2-(benzylthio)ethylene)glycine ethyl ester;

[0091] 8) N^(α)-(2-(benzylthio)ethylene)(S)leucine methyl ester;

[0092] 9) N^(α)-(3-(benzylthio)propylene)(S)leucine methyl ester:

[0093] 10) Boc-N^(α)-(2-(benzylthio)ethylene)glycine;

[0094] 11) Boc-N^(α)-(2-(benzylthio)ethylene)(S)phenylalanine;

[0095] 12) Boc-N^(α)-(3-(benzylthio)propylene)(S)phenylalanine;

[0096] 13)Boc-L-phenylalanyl-N^(α)-(2-(benzylthio)ethylene)glycine-ethyl ester;

[0097] 14)Boc-L-phenylalanyl-N^(α)-(2-(benzylthio)ethylene)-(S)phenylalaninemethyl ester;

[0098] 15) N^(α)(Fmoc)-(2-t-butyl carboxy ethylene)glycine;

[0099] 16) N^(α)(Fmoc)-(3-t-butyl carboxy propylene)glycine;

[0100] 17) N^(α)(Fmoc)(2-t-butyl carboxy ethylene)(S)phenylalanine;

[0101] 18) N^(α)(Fmoc)(2-Boc amino ethylene)glycine;

[0102] 19) N^(α)(Fmoc)(3-Boc amino propylene)glycine;

[0103] 20) N^(α)(Fmoc)(4-Boc amino butylene)glycine; and

[0104] 21) N^(α)(Fmoc)(6-Boc amino hexylene)glycine.

[0105] Novel, practical, generally applicable processes for thepreparation of these N^(α)-ω-functionalized derivatives of amino acidsare a further aspect of this invention.

[0106] As such, an object of this invention is a method of making anω-functionalized amino acid derivative of the general Formula:

[0107] wherein X is a spacer group selected from the group consisting ofalkylene, substituted alkylene, arylene, cycloalkylene and substitutedcycloalkylene; R is the side chain of an amino acid, such as H, CH₃,etc.; A and B are protecting groups selected from the group consistingof alkyloxy, substituted alkyloxy, or aryloxy carbonyls;

[0108] comprising the steps of:

[0109] i) reacting a diamine compound of the general Formula:

[0110]  wherein A, B and X are as defined above,

[0111] with a triflate of Formula CF₃SO₂—O—CH(R)—CO—E wherein E is acarboxyl protecting group and R is as defined above; to yield a compoundof Formula:

[0112] wherein A, B, E, R and X are as defined above

[0113] ii) and deprotecting the carboxyl to yield anN^(α)ω-functionalized amino acid derivative, wherein the ω-functionalgroup is an amine.

[0114] A further object of this invention is a method of making anω-functionalized amino acid derivative of the general Formula:

[0115] where B is a protecting group selected from the group ofsubstituted alkyloxy, substituted alkyloxy, or aryloxy carbonyls; R isthe side chain of an amino acid, such as H, CH₃, etc.; X is a spacergroup selected from the group of alkylene, substituted alkylene,arylene, cycloalkylene or substituted cycloalkylene; and A is aprotecting group selected from the group of alkyl or substituted alkyl,thio ethers or aryl or substituted aryl thio ethers;

[0116] comprising the steps of:

[0117] i) reacting a compound of the general Formula B—NH—X—S—A with atriflate of the general Formula CF₃SO₂—O—CH(R)—CO—E wherein E is acarboxyl protecting group and A, X and R are as defined above, to give acompound of the Formula:

[0118] ii) selectively removing the protecting group E, and

[0119] iii) protecting the free amino group to yield anN^(α)(ω-functionalized) amino acid derivative, wherein the ω-functionalgroup is a thiol.

[0120] A further object of this invention is a method of making anω-functionalized amino acid derivative of the general Formula:

[0121] where B is a protecting group selected from the group ofalkyloxy, substituted alkyloxy, or aryloxy carbonyls; R is the sidechain of an amino acid, such as H, CH₃, etc.; X is a spacer groupselected from the group of alkylene, substituted alkylene, arylene,cycloalkylene or substituted cycloalkylene; and A is a protecting groupselected from the group of alkyl or substituted alkyl, esters, or thioesters or substituted aryl esters or thio esters;

[0122] comprising the steps of:

[0123] i) reacting a compound of the general Formula B—NH—X—CO—A with atriflate of the general Formula CF₃SO₂—O—CH(R)—CO—E wherein E is acarboxyl protecting group and A, B, X and R are as defined above, togive a compound of Formula:

[0124] ii) and selectively removing protecting group E, to yield anN^(α)(ω-functionalized) amino acid derivative, wherein the ω-functionalgroup is a carboxyl.

[0125] A further aspect of this invention is to provide methods for thepreparation of novel backbone cyclic peptides, comprising the steps ofincorporating at least one N^(α)-ω-functionalized derivatives of aminoacids into a peptide sequence and subsequently selectively cyclizing thefunctional group with one of the side chains of the amino acids in saidpeptide sequence, or with another ω-functionalized amino acidderivative.

[0126] Backbone cyclized analogs of the present invention may be used aspharmaceutical compositions and for methods for the treatment ofdisorders including: acute asthma, septic shock, brain trauma and othertraumatic injury, post-surgical pain, all types of inflammation,cancers, endocrine disorders and gastrointestinal disorders.

[0127] Therefore, further objects of the present invention are directedto pharmaceutical compositions comprising pharmacologically activebackbone cyclized peptide agonists and antagonists prepared according tothe methods disclosed herein and a pharmaceutically acceptable carrieror diluent; and methods for the treatment of inflammation, septic shock,cancer or endocrine disorders and gastrointestinal disorders therewith.

BRIEF DESCRIPTION OF THE DRAWINGS

[0128]FIG. 1 is a graph showing in vitro biostability of somatostatinand three analogs thereof in human serum. The graph depicts thepercentage of undegraded molecules for each of the compounds initiallyand after various periods of time.

DETAILED DESCRIPTION OF THE INVENTION

[0129] Definitions

[0130] All abbreviations used are in accordance with the IUPAC-IUBPrecommendations on Biochemical Nomenclature (J. Biol. Chem.,247:977-983, 1972) and later supplements.

[0131] As used herein and in the claims, the phrase “an amino acid sidechain” refers to the distinguishing substituent attached to the α-carbonof an amino acid; such distinguishing groups are well known to thoseskilled in the art. For instance, for the amino acid glycine, the Rgroup is H; for the amino acid alanine, R is CH₃, and so on. Othertypical side chains of amino acids include the groups: (CH₃)₂CH—,(CH₃)₂CHCH₂—, CH₃CH₂CH(CH₃)—, CH₃S(CH₂)₂—, HOCH₂—, CH₃CH(OH)—, HSCH₂—,NH₂C (═O)CH₂—, NH₂C(═O)(CH₂)₂—, NH₂(CH₂)₃—, HOC(═O)CH₂—HOC(═O)(CH₂)₂—,NH₂(CH₂)₄—, C(NH₂)₂ NH(CH₂)₃—, HO-phenyl-CH₂—, benzyl, methylindole, andmethylimidazole.

[0132] As used herein and in the claims, the letters “(AA)” and the term“amino acid” are intended to include common natural or synthetic aminoacids, and common derivatives thereof, known to those skilled in theart, including but not limited to the following. Typical amino-acidsymbols denote the L configuration unless otherwise indicated by Dappearing before the symbol. Abbreviated Designation Amino Acids Abuα-Amino butyric acid Ala L-Alanine Arg L-Arginine Asn L-Asparagine AspL-Aspartic acid βAsp(Ind) β-Indolinyl aspartic acid Cys L-Cysteine GluL-Glutamic acid Gln L-Glutamine Gly Glycine His L-Histidine Hyptrans-4-L-Hydroxy Proline Ile L-Isoleucine Leu L-Leucine Lys L-LysineMet L-Methionine Nal β-Naphthyl alanine Orn Ornithine PheL-Phenylalanine Pro L-Proline Ser L-Serine Thr L-Threonine TrpL-Tryptophane Tyr L-Tyrosine Val L-Valine

[0133] Typical protecting groups, coupling agents, reagents and solventssuch as but not limited to those listed below have the followingabbreviations as used herein and in the claims. One skill in the artwould understand that the compounds listed within each group may be usedinterchangeably; for instance, a compound listed under “reagents andsolvents” may be used as a protecting group, and so on. Further, oneskill in the art would know other possible protecting groups, couplingagents and reagents/solvents; these are intended to be within the scopeof this invention. Abbreviated Designation Protecting Groups AdaAdamantane acetyl Alloc Allyloxycarbonyl Allyl Allyl ester Boctert-butyloxycarbonyl Bzl Benzyl Fmoc Fluorenylmethyloxycarbonyl OBzlBenzyl ester OEt Ethyl ester OMe Methyl ester Tos (Tosyl)p-Toluenesulfonyl Trt Triphenylmethyl Z Benzyloxycarbonyl

[0134] Abbreviated Designation Coupling Agents BOPBenzotriazol-1-yloxytris- (dimethyl-amino) phosphoniumhexafluorophosphate DIC Diisopropylcarbodiimide HBTU2-(1H-Benzotriazol-1-yl)- 1,1,3,3-tetramethyluronium hexafluorophosphatePyBrOP Bromotripyrrolidinophosphonium hexafluorophosphate PyBOPBenzotriazol-1-yl-oxy-tris- pyrrolidino-phosphonium hexafluorophosphateTBTU O-(1,2-dihydro-2-oxo-1-pyridyl)- N,N,N′,N′-tetramethyluroniumtetrafluoroborate

[0135] Abbreviated Designation Reagents and Solvents ACN AcetonitrileAcOH Acetic acid Ac₂O Acetic acid anhydride AdacOH Adamantane aceticacid Alloc-Cl Allyloxycarbonyl chloride Boc₂O Di-tert butyl dicarbonateDMA Dimethylacetamide DMF N,N-dimethylformamide DIEADiisopropylethylamine Et₃N Triethylamine

[0136] or more triple carbon-carbon bonds which may occur in any stablepoint along the chain, such as ethynyl, propynyl, and the like.

[0137] As used herein and in the claims, “aryl” is intended to mean anystable 5- to 7-membered monocyclic or bicyclic or 7-to 14-memberedbicyclic or tricyclic carbon ring, any of which may be saturated,partially unsaturated or aromatic, for example, phenyl, naphthyl,indanyl, or tetrahydronaphthyl tetralin, etc.

[0138] As used herein and in the claims, “alkyl halide” is intended toinclude both branched and straight-chain saturated aliphatic hydrocarbongroups having one to ten carbon atoms, wherein 1 to 3 hydrogen atomshave been replaced by a halogen atom such as Cl, F, Br, and I.

[0139] As used herein and in the claims, the term “heterocyclic” isintended to mean any stable 5- to 7-membered monocyclic or bicyclic or7- to 10-membered bicyclic heterocyclic ring, which is either saturatedor unsaturated, and which consists of carbon atoms and from 1 to 3heteroatoms selected from the group consisting of N, O and S and whereinthe nitrogen and sulfur atoms may optionally be oxidized, and thenitrogen atom optionally be quaternized, and including any bicyclicgroup in which any of the above-defined heterocyclic rings is fused to abenzene ring. The heterocyclic ring may be attached to its pendant groupat any heteroatom or carbon atom which results in a stable structure.The heterocyclic rings described herein may be substituted on carbon oron a nitrogen atom if the resulting compound is stable. Examples of suchheterocycles include, but are not limited to pyridyl, pyrimidinyl,furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl,benzofuranyl, benzothiophenyl, indolyl, indolenyl, quinolinyl,piperidonyl, pyrrolidinyl, pyrrolinyl, tetrahydrofuranyl,tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oroctahydroisoquinolinyl and the like.

[0140] As used herein and in the claims, the phrase “therapeuticallyeffective amount” means that amount of novel EtOAc Ethyl acetate FmocOSu9-fluorenylmethyloxy carbonyl N-hydroxysuccinimide ester HOBT1-Hydroxybenzotriazole HF Hydrofluoric acid MeOH Methanol Mes (Mesyl)Methanesulfonyl NMP 1-methyl-2-pyrrolidinone nin. Ninhydrin i-PrOHIso-propanol Pip Piperidine PP 4-pyrrolidinopyridine Pyr Pyridine SRIFSomatotropin release inhibiting factor SST Somatostatin SSTRSomatostatin receptor TEA Triethylamine TFA Trifluoroacetic acid THFTetrahydrofuran Triflate (Trf) Trifluoromethanesulfonyl Trf₂OTrifluoromethanesulfonic acid anhydride

[0141] The compounds herein described may have asymmetric centers. Allchiral, diastereomeric, and racemic forms are included in the presentinvention. Many geometric isomers of olefins and the like can also bepresent in the compounds described herein, and all such stable isomersare contemplated in the present invention.

[0142] By “stable compound” or “stable structure” is meant herein acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and Formulation into anefficacious therapeutic agent.

[0143] As used herein and in the claims, “alkyl” or “alkylenyl” isintended to include both branched and straight-chain saturated aliphatichydrocarbon groups having one to ten carbon atoms; “alkenyl” is intendedto include hydrocarbon chains of either a straight or branchedconfiguration and one or more unsaturated carbon-carbon bonds which mayoccur in any stable point along the chain, such as ethenyl, propenyl,and the like; and “alkynyl” is intended to include hydrocarbon chains ofeither a straight or branched configuration and one backbone cyclizedpeptide analog or composition comprising same to administer to a host toachieve the desired results for the indications described herein, suchas but not limited of inflammation, septic shock, cancer, endocrinedisorders and gastrointestinal disorders.

[0144] The term, “substituted” as used herein and in the claims, meansthat any one or more hydrogen atoms on the designated atom is replacedwith a selection from the indicated group, provided that the designatedatom's normal valency is not exceeded, and that the substitution resultsin a stable compound.

[0145] When any variable (for example R, x, z, etc.) occurs more thanone time in any constituent or in Formulae (I to XX) or any otherFormula herein, its definition on each occurrence is independent of itsdefinition at every other occurrence. Also, combinations of substituentsand/or variables are permissible only if such combinations result instable compounds.

[0146] Synthetic Approach

[0147] According to the present invention peptide analogs are cyclizedvia bridging groups attached to the alpha nitrogens of amino acids thatpermit novel non-peptidic linkages. In general, the procedures utilizedto construct such peptide analogs from their building units rely on theknown principles of peptide synthesis; most conveniently, the procedurescan be performed according to the known principles of solid phasepeptide synthesis. The innovation requires replacement of one or more ofthe amino acids in a peptide sequence by novel building units of thegeneral Formula:

[0148] wherein R is the side chain of an amino acid, X is a spacer groupand G is the functional end group by means of which cyclization will beeffected. The side chain R is the side chain of any natural or syntheticamino acid that is selected to be incorporated into the peptide sequenceof choice. X is a spacer group that is selected to provide a greater orlesser degree of flexibility in order to achieve the appropriateconformational constraints of the peptide analog. Such spacer groupsinclude alkylene chains, substituted, branched and unsaturatedalkylenes, arylenes, cycloalkylenes, unsaturated and substitutedcycloakylenes. Furthermore, X and R can be combined to form aheterocyclic structure.

[0149] A preferred embodiment of the present invention utilizes alkylenechains containing from two to ten carbon atoms.

[0150] The terminal (ω) functional groups to be used for cyclization ofthe peptide analog include but are not limited to:

[0151] a. Amines, for reaction with electrophiles such as activatedcarboxyl groups, aldehydes and ketones (with or without subsequentreduction), and alkyl or substituted alkyl halides.

[0152] b. Alcohols, for reaction with electrophiles such as activatedcarboxyl groups.

[0153] c. Thiols, for the formation of disulfide bonds and reaction withelectrophiles such as activated carboxyl groups, and alkyl orsubstituted alkyl halides.

[0154] d. 1,2 and 1,3 Diols, for the formation of acetals and ketals.

[0155] e. Alkynes or Substituted Alkynes, for reaction with nucleophilessuch as amines, thiols or carbanions; free radicals; electrophiles suchas aldehydes and ketones, and alkyl or substituted alkyl halides; ororganometallic complexes.

[0156] f. Carboxylic Acids and Esters, for reaction with nucleophiles(with or without prior activation), such as amines, alcohols, andthiols.

[0157] g. Alkyl or Substituted Alkyl Halides or Esters, for reactionwith nucleophiles such as amines, alcohols, thiols, and carbanions (fromactive methylene groups such as acetoacetates or majonates); andformation of free radicals for subsequent reaction with alkenes orsubstituted alkenes, and alkynes or substituted alkynes.

[0158] h. Alkyl or Aryl Aldehydes and Ketones for reaction withnucleophiles such as amines (with or without subsequent reduction),carbanions (from active methylene groups such as acetoacetates ormalonates), diols (for the formation of acetals and ketals).

[0159] i. Alkenes or Substituted Alkenes, for reaction with nucleophilessuch as amines, thiols, carbanions, free radicals, or organometalliccomplexes.

[0160] j. Active Methylene Groups, such as malonate esters, acetoacetateesters, and others for reaction with electrophiles such as aldehydes andketones, alkyl or substituted alkyl halides.

[0161] It will be appreciated that during synthesis of the peptide thesereactive end groups, as well as any reactive side chains, must beprotected by suitable protecting groups. Suitable protecting groups foramines are alkyloxy, substituted alkyloxy, and aryloxy carbonylsincluding, but not limited to, tert butyloxycarbonyl (Boc),Fluorenylmethyloxycarbonyl (Fmoc), Allyloxycarbonyl (Alloc) andBenzyloxycarbonyl (Z).

[0162] Carboxylic end groups for cyclizations may be protected as theiralkyl or substituted alkyl esters or thio esters or aryl or substitutedaryl esters or thio esters. Examples include but are not limited totertiary butyl ester, allyl ester, benzyl ester, 2-(trimethylsilyl)ethylester and 9-methyl fluorenyl.

[0163] Thiol groups for cyclizations may be protected as their alkyl orsubstituted alkyl thio ethers or disulfides or aryl or substituted arylthio ethers or disulfides. Examples of such groups include but are notlimited to tertiary butyl, trityl(triphenylmethyl), benzyl,2-(trimethylsilyl)ethyl, pixyl(9-phenylxanthen-9-yl), acetamidomethyl,carboxy-methyl, 2-thio-4-nitropyridyl.

[0164] It will further be appreciated by the artisan that the variousreactive moieties will be protected by different protecting groups toallow their selective removal. Thus, a particular amino acid will becoupled to its neighbor in the peptide sequence when the N^(α)isprotected by, for instance, protecting group A. If an amine is to beused as an end group for cyclization in the reaction scheme the N^(ω)will be protected by protecting group B, or an E amino group of anylysine in the sequence will be protected by protecting group C, and soon.

[0165] The coupling of the amino acids to one another is performed as aseries of reactions as is known in the art of peptide synthesis. Novelbuilding units of the invention, namely the N^(α)-ω functionalized aminoacid derivatives are incorporated into the peptide sequence to replaceone or more of the amino acids. If only one such N^(α)-ω functionalizedamino acid derivative is selected, it will be cyclized to a side chainof another amino acid in the sequence. For instance: (a) anN^(α)-(ω-amino alkylene) amino acid can be linked to the carboxyl groupof an aspartic or glutamic acid residue; (b) an N^(α)-(ω-carboxylicalkylene) amino acid can be linked to the ε-amino group of a lysineresidue; (c) an N^(α)-(ω-thio alkylene) amino acid can be linked to thethiol group of a cysteine residue; and so on. A more preferredembodiment of the invention incorporates two such N^(α)-ω-functionalizedamino acid derivatives which may be linked to one another to formN-backbone to N-backbone cyclic peptide analogs. Three or more suchbuilding units can be incorporated into a peptide sequence to createbi-cyclic peptide analogs as will be elaborated below. Thus, peptideanalogs can be constructed with two or more cyclizations, includingN-backbone to N-backbone, as well as backbone to side-chain or any otherpeptide cyclization.

[0166] As stated above, the procedures utilized to construct peptideanalogs of the present invention from novel building units generallyrely on the known principles of peptide synthesis. However, it will beappreciated that accommodation of the procedures to the bulkier buildingunits of the present invention may be required. Coupling of the aminoacids in solid phase peptide chemistry can be achieved by means of acoupling agent such as but not limited to dicyclohexycarbodiimide (DCC),bis(2-oxo-3-oxazolidinyl) phosphinic chloride (BOP-Cl),benzotriazolyl-N-oxytrisdimethyl-aminophosphonium hexafluoro phosphate(BOP), 1-oxo-1-chlorophospholane (Cpt-Cl), hydroxybenzotriazole (HOBT),or mixtures thereof.

[0167] It has now been found that coupling of the bulky building unitsof the present invention may require the use of additional couplingreagents including, but not limited to: coupling reagents such as PyBOP®(Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate), PyBrOP® (Bromo-tris-pyrrolidino-phosphoniumhexafluoro-phosphate), HBTU(2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluoro-phosphate), TBTU(2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate).

[0168] Novel coupling chemistries may be used, such as pre-formedurethane-protected N-carboxy anhydrides (UNCA's) and pre-formed acylfluorides. Said coupling may take place at room temperature and also atelevated temperatures, in solvents such as toluene, DCM(dichloromethane), DMF (dimethylformamide), DMA (dimethylacetamide), NMP(N-methyl pyrrolidinone) or mixtures of the above.

[0169] One object of the present invention is a method for thepreparation of backbone cyclized peptide analogs of Formula (I):

[0170] wherein the substituents are as defined above;

[0171] comprising the steps of incorporating at least oneN^(α)-ω-functionalized derivatives of amino acids of Formula (VI):

[0172] wherein X is a spacer group selected from the group consisting ofalkylene, substituted alkylene, arylene, cycloalkylene and substitutedcycloalkylene; R′ is an amino acid side chain such as H, CH₃, etc.,optionally bound with a specific protecting group; B is a protectinggroup selected from the group consisting of alkyloxy, substitutedalkyloxy, or aryloxy carbonyls; and G is a functional group selectedfrom the group consisting of amines, thiols, alcohols, carboxylic acidsand esters, aldehydes, alcohols and alkyl halides; and A is a specificprotecting group of G;

[0173] with a compound of the Formula (VII):

H₂N—(AA)_(f)—CO—E  Formula (VII)

[0174] wherein f is an integer from 1 to 10; (AA) designates an aminoacid residue wherein the amino acid residues may be the same ordifferent, and E is a hydroxyl, a carboxyl protecting group or an amideto give a compound of the general Formula:

[0175] (ii) selectively removing protecting group B and reacting theunprotected compound with a compound of Formula:

B—NH—(AA)_(e)—COOH  Formula (IX)

[0176] wherein B and (AA) are as described above and e is an integerfrom 1 to 10,

[0177] to give a compound of Formula:

[0178] wherein B, (AA), e, R¹, and f are as described above;

[0179] (iii) removing the protecting group B from the compound of theFormula (X) and reacting the unprotected compound with a compound ofFormula:

[0180] wherein X′ is a spacer group selected from the group consistingof alkylenes, substituted alkylenes, arylenes, cycloalkylenes andsubstituted alkylenes; G′ is a functional group selected from amines,thiols, carboxyls, aldehydes or alcohols; A′ is a specific-protectinggroup thereof; R¹ is an amino acid side chain such as H, CH₃, etc.,optionally bound with a specific protecting group; and B is a protectinggroup;

[0181] to yield a compound of Formula:

[0182] (iv) removing the protecting group B and reacting the unprotectedcompound with a compound of Formula:

B—NH—(AA)_(d)—COOH  Formula (IXa)

[0183] to yield a compound of Formula:

[0184] (v) selectively removing protecting groups A and A′ and reactingthe terminal groups G and G′ to form a compound of the Formula:

[0185] wherein d, e and f are independently an integer from 1 to 10;(AA) is an amino acid residue wherein the amino acid residues in eachchain may be the same or different; E is an hydroxyl group, a carboxylprotecting group or an amino group; R and R′ are independently an aminoacid side-chain such as H, CH₃, etc.; and the line designates a bridginggroup of the Formula: —X—M—Y—W—Z—

[0186] wherein M and W are independently selected from the groupconsisting of disulfide, amide, thioether, imine, ether, and alkene; X,Y and Z are independently selected from the group consisting ofalkylene, substituted alkylene, arylene, cycloalkylene, and substitutedcycloalkylene;

[0187] (vi) removing all remaining protecting groups to yield a compoundof Formula (I).

[0188] Bicyclic analogs are prepared in the same manner, that is, byrepetition of steps (v) and (vi). The determination of which residuesare cyclized with which other residues is made through the choice ofblocking groups. The various blocking groups may be removed selectively,thereby exposing the selected reactive groups for cyclization.

[0189] Preferred are methods for the preparation of backbone cyclizedpeptide analogs of Formula (I) wherein G is an amine, thiol or carboxylgroup; R and R¹ are each other than H, such as CH₃, (CH₃)₂CH—,(CH₃)₂CHCH₂—, CH₃CH₂CH(CH₃) —, CH₃S(CH₂)₂—, HOCH₂—, CH₃CH(OH)—, HSCH₂—,NH₂C(═O)CH₂—, NH₂C(═O)(CH₂)₂—, HOC(═O)CH₂—, HOC(═O)(CH₂)₂—, NH₂ (CH₂)₄—,C(NH₂)₂ NH(CH₂)₃—, HO-phenyl-CH₂—, benzyl, methylindole, andmethylimidazole, and wherein E is covalently bound to an insolublepolymeric support.

[0190] Another object of the present invention is a method for thepreparation of backbone cyclized peptide analogs of Formula (II):

[0191] wherein the substituents are as defined above;

[0192] comprising the steps of: incorporating at least oneω-functionalized amino acid derivative of the general Formula (VI):

[0193] wherein X is a spacer group selected from the group consisting ofalkylene, substituted alkylene, arylene, cycloalkylene and substitutedcycloalkylene; R is the side chain of an amino acid, such as H, CH₃,etc.; B is a protecting group selected from the group consisting ofalkyloxy, substituted alkyloxy, or aryloxy carbonyls; and G is afunctional group selected from the group consisting of amines, thiols,alcohols, carboxylic acids and esters or alkyl halides and A is aprotecting group thereof;

[0194] into a peptide sequence and subsequently selectively cyclizingthe functional group with one of the side chains of the amino acids insaid peptide sequence.

[0195] Preferred is the method for the preparation of backbone cyclizedpeptide analogs of Formula (II) wherein G is a carboxyl group or a thiolgroup; R is CH₃, (CH₃)₂CH—, (CH₃)₂CHCH₂—, CH₃CH₂CH (CH₃)—, CH₃S (CH₂)₂—,HOCH₂—, CH₃CH(OH)—, HSCH₂—, NH₂C (═O) CH₂—, NH₂C (═O)(CH₂)₂—,HOC(═O)CH₂—, HOC(═O)(CH₂)₂—, NH₂ (CH₂)₄—, C(NH₂)₂ NH(CH₂)₃—,HO-phenyl-CH₂—, benzyl, methylindole, and methylimidazole, and wherein Eis covalently bound to an insoluble polymeric support.

[0196] Preparation of backbone to side chain cyclized peptide analogs isexemplified in Scheme I below. In this schematic example, the bridginggroup consists of alkylene spacers and an amide bond formed between anacidic amino acid side chain (e.g. aspartic or glutamic acid) and anω-functionalized amino acid having a terminal amine.

Scheme I Preparation of Peptides with Backbone to Side Chain Cyclization

[0197] One preferred procedure for preparing the desired backbone cyclicpeptides involves the stepwise synthesis of the linear peptides on asolid support and the backbone cyclization of the peptide either on thesolid support or after removal from the support. The C-terminal aminoacid is bound covalently to an insoluble polymeric support by acarboxylic acid ester or other linkages such as amides. An example ofsuch support is a polystyrene-co-divinyl benzene resin. The polymericsupports used are those compatible with such chemistries as Fmoc and Bocand include for example PAM resin, HMP resin and chloromethylated resin.The resin bound amino acid is deprotected for example with TFA to give(1) below and to it is coupled the second amino acid, protected on theN^(α) for example by Fmoc, using a coupling reagent like BOP. The secondamino acid is deprotected to give (3) using for example piperidine 20%in DMF. The subsequent protected amino acids can then be coupled anddeprotected at ambient temperature. After several cycles of coupling anddeprotection that gives peptide (4), an amino acid having for examplecarboxy side chain is coupled to the desired peptide. One such aminoacid is Fmoc-aspartic acid t-butyl ester. After deprotection of theN^(α) Fmoc protecting group that gives peptide (5), the peptide is againelongated by methods well known in the art to give (6). Afterdeprotection a building unit for backbone cyclization (the preparationof which is described in Schemes III-VIII) is coupled to the peptideresin using for example the coupling reagent BOP to give (7). One suchbuilding unit is for example Fmoc-N^(α)(ω-Boc-amino alkylene)amino acid.After deprotection the peptide can then be elongated, to the desiredlength using methods well known in the art to give (8). The coupling ofthe protected amino acid subsequent to the building unit is performed bysuch coupling agents exemplified by PyBrOP® to ensure high yield.

[0198] After the linear, resin bound peptide, e.g. (8), has beenprepared the ω-alkylene-protecting groups for example Boc and t-Bu areremoved by mild acid such as TFA to give (9). The resin bound peptide isthen divided into several parts. One part is subjected to on-resincyclization using for example TBTU as cyclization agent in DMF to ensurehigh yield of cyclization, to give the N-backbone to side chain cyclicpeptide resin (10). After cyclization on the resin the terminal aminoprotecting group is removed by agents such as piperidine and thebackbone to side chain cyclic peptide (11) is obtained after treatmentwith strong acid such as HF. Alternatively, prior to the removal of thebackbone cyclic peptide from the resin, the terminal amino group isblocked by acylation with agents such as acetic anhydride, benzoicanhydride or any other acid such as adamantyl carboxylic acid activatedby coupling agents such as BOP.

[0199] The other part of the peptide-resin (9) undergoes protecting ofthe side chains used for cyclization, for example the ω-amino andcarboxy groups. This is done by reacting the ω-amino group with forexample Ac₂O and DMAP in DMF and activating the free ω-carboxy group byfor example DIC and HOBT to give the active ester which is then reactedwith for example Dh₃NH₂ to give the linear analog (13) of the cyclicpeptide (10). Removal of the peptide from the resin and subsequentremoval of the side chains protecting groups by strong acid such as HFto gives (14) which is the linear analog of the backbone to side chaincyclic peptide (11).

[0200] The linear analogs are used as reference compounds for thebiological activity of their corresponding cyclic compounds.

[0201] [Reaction Scheme I follows at this point]

[0202] The selection of N^(α) and side chain protecting groups is, inpart, dictated by the cyclization reaction which is done on thepeptide-resin and by the procedure of removal of the peptide from theresin. The N^(α) protecting groups are chosen in such a manner thattheir removal will not effect the removal of the protecting groups ofthe N^(α)(ω-aminoalkylene) protecting groups. In addition, the removalof the N^(α)(ω-aminoalkylene)protecting groups or any other protectinggroups on ω-functional groups prior to the cyclization, will not effectthe other side chain protection and/or the removal of the peptide fromthe resin. The selection of the side chain protecting groups other thanthose used for cyclization is chosen in such a manner that they can beremoved subsequently with the removal of the peptide from the resin.Protecting groups ordinarily employed include those which are well knownin the art, for example, urethane protecting substituents such as Fmoc,Boc, Alloc, Z and the like.

[0203] It is preferred to utilize Fmoc for protecting the α-amino groupof the amino acid undergoing the coupling reaction at the carboxyl endof said amino acid. The Fmoc protecting group is readily removedfollowing such coupling reaction and prior to the subsequent step by themild action of base such as piperidine in DMF. It is preferred toutilize Boc for protecting ω-amino group of the N^(α)(ω-aminoalkylene)group and t-Bu for protecting the carboxy group of the amino acidsundergoing the reaction of backbone cyclization. The Boc and t-Buprotecting groups are readily removed simultaneously prior to thecyclization.

Scheme II Preparation of Peptides with Backbone to Backbone Cyclization

[0204] Preparation of N-backbone to N-backbone cyclized peptide analogsis exemplified in scheme II. In this schematic example, the buildinggroup consists of alkylene spacers and two amide bonds.

[0205] A building unit for backbone cyclization (the preparation ofwhich described in Schemes III-VIII) is coupled to a peptide resin, forexample peptide-resin (4), using for example the coupling reagent BOP togive (16). One such building unit is for example Fmoc-Nα(ω-Boc-aminoalkylene)amino acid. The side chain Boc protecting group is removed bymild acid such as TFA in DCM and an N-Boc protected ω-amino acid, or anyother Boc protected amino acid, is coupled to the side chain amino groupusing coupling agent such as BOP to give peptide-resin (17).

[0206] After deprotection of the N^(α) Fmoc protecting group by mildbase such as piperidine in DMF, the peptide can then be elongated, ifrequired, to the desired length using methods well known in the art togive (18). Alternatively, the deprotection of the N^(α) Fmoc andsubsequent elongation of the peptide can be done before deprotection ofthe side chain Boc protecting group. The elongation of the N-alkyleneside chain allow control of the ring size. The coupling of the protectedamino acid subsequent to the building unit is performed by such couplingagents exemplified by PyBrOP® to ensure high yield.

[0207] After deprotection of the terminal N^(α) Fmoc group, a secondbuilding unit, for example Fmoc-N^(α)(ω-t-Bu-carboxy-alkylene)amino acidis coupled to the peptide-resin to give (19). After deprotection of theN^(α) Fmoc protecting group, the peptide can then be elongated, ifrequired, to the desired length using methods well known in the art togive (20). The coupling of the protected amino acid subsequent to thebuilding unit is performed by such coupling agents exemplified byPyBrOP® to ensure high yield. After the linear, resin bound peptide,e.g. (20), has been prepared the ω-alkylene-protecting groups forexample Boc and t-Bu are removed by mild acid such as TFA to give (21).The resin peptide is then divided into several parts. One part issubjected to on-resin cyclization using for example TBTU as cyclizationagent in DMF to ensure high yield of cyclization, to give the N-backboneto N-backbone cyclic peptide resin (22). After cyclization on the resinthe terminal amino protecting group is removed by agents such aspiperidine and the backbone to backbone cyclic peptide (23) is obtainedafter treatment with strong acid such as HF. Alternatively, prior to theremoval of the backbone cyclic peptide from the resin, the terminalamino group of (22) is blocked, after deprotection, by acylation withagents such as acetic anhydride, benzoic anhydride or any other acidsuch as adamantyl carboxylic acid activated by coupling agents such asBOP to give the N-terminal blocked backbone to backbone cyclic peptide(24).

[0208] The other part of the peptide-resin (21) undergoes protecting ofthe side chains used for cyclization, for example the ω-amino andcarboxy groups. This is done by reacting the ω-amino group with forexample AC₂O and DMAP in DMF and activating the free ω-carboxy group byfor example DIC and HOBT to give the active ester which is then reactedwith for example MeNH₂. Removal of the peptide from the resin andsubsequent removal of the side chains protecting groups by strong acidsuch as HF to gives (26) which is the linear analog of the backbone tobackbone cyclic peptide (23). The linear analogs are used as referencecompounds for the biological activity of their corresponding cycliccompounds.

[0209] [Reaction Scheme II follows at this point]

[0210] Novel Synthesis of Building Units

[0211] The novel synthesis providing N(ω-(functionalized) alkylene)amino acids used to generate backbone cyclic peptides is depicted inschemes III-VIII. In this approach we have implemented the followingchanges in order to devise a practical, general synthesis:

[0212] 1. The nucleophile is a secondary nitrogen, which is a betternucleophile than the primary nitrogen previously used. This alsoprevents the possibility of double alkylation.

[0213] 2. The leaving group was changed to trifluoromethanesulfonyl(triflate), which has a much lower tendency to eliminate than a halogen,thus making it possible to implement the synthesis with amino acidsother than glycine. Furthermore, the triflate leaving group preventsracemization during the alkylation reaction.

[0214] 3. The carboxylate is esterified prior to the substitutionreaction, to facilitate the substitution by removing the negative chargenext to the electrophilic carbon.

Scheme III Preparation of N^(α), N^(ω) Protected ω-amino Alkylene AminoAcids Building Units

[0215] One preferred procedure for the preparation of protectedN^(α)(ω-amino alkylene) amino acids involves the N^(α) alkylation ofsuitably protected diamino alkanes. One preferred N^(α), N^(ω) di-protected diamino alkane is for example N^(α)-Benzyl, N^(ω)-Boc diaminoalkane (27). This starting material contains one protecting group suchas Boc which is necessary for the final product, and a temporaryprotecting group such as Bzl to minimize unwanted side reactions duringthe preparation of the titled compound. One preferred procedure for thepreparation of the starting material (27) involves reductive alkylationof N-Boc diamino alkane with aldehydes such as benzaldehyde. Thetemporary protection of the N^(α) amino group, which is alkylated in thereaction by such protecting groups as Bzl, minimizes the dialkylationside reaction and allows removal by such conditions that do not removethe N^(ω)-protecting group.

[0216] The N^(α),N^(ω) di-protected diamino alkane is reacted with forexample chiral α-hydroxy α-substituted acid esters where the hydroxylmoiety is converted to a leaving group for example Triflate.

[0217] The use of Triflate as the leaving group was found to be superiorto other leaving groups such as halogens, Tosyl, Mesyl, etc., because itprevents the β-elimination reaction encountered with the other leavinggroups. The use of Triflate as the leaving group also ensures highoptical purity of the product (28). The temporary N^(α) protectinggroup, such as Bzl, and the carboxyl protecting group, such as methylester, are removed by mild conditions, such as catalytic hydrogenationand hydrolysis, that do not remove the N^(ω) protecting group such asBoc to give the N^(ω) protected amino acid (29). Introduction of theN^(α) protecting group suitable for peptide synthesis is accomplished bymethods well known in the art, to give the protected N^(α)(N^(ω)protected amino alkylene) amino acid (30).

[0218] The choice of the N^(α) and the N^(ω) protecting groups isdictated by the use of the building units in peptide synthesis. Theprotecting groups have to be orthogonal to each other and orthogonal tothe other side chains protecting groups in the peptide. Combinations ofN^(α) and N^(ω) protecting groups are for example: N^(α)-Fmoc,N^(ω)-Boc; N^(α)-Fmoc, N^(ω)-Alloc; N^(α)-Boc, N^(ω)-Alloc. Thesecombinations are suitable for peptide synthesis and backbonecyclization, either on solid support or in solution.

Scheme IV Preparation of N^(α), N^(ω) Protected ω-amino Alkylene GlycineBuilding Units

[0219] One preferred procedure for the preparation of protectedN^(α)(ω-amino alkylene)glycines involves the reaction of the N^(α),N^(ω) di-protected di amino alkane (27) with commercially availableα-activated carboxylic acid esters, for example benzylbromo acetate.Since the titled compound is achiral, the use of leaving groups such asTrf, Tos or Mes is not necessary. The use of the same temporaryprotecting groups for the N^(α) and the carboxy groups, for example theBzl protecting group, ensures the prevention of the undesireddialkylation side reaction and allows concomitant removal of thetemporary protecting groups thus giving high yield of the N^(ω)protected amino acid (32). Introduction of the N^(α) protecting groupsuitable for peptide synthesis is accomplished by methods well known inthe art, to give the protected N^(α)(N^(ω) protected aminoalkylene)glycines (33).

[0220] The choice of the N^(α) and the N^(ω) protecting groups isdictated by the use of the building units in peptide synthesis. Theprotecting groups have to be orthogonal to each other and orthogonal tothe other side chains protecting groups in the peptide. Combinations ofN^(α) and N^(ω) protecting groups are for example: N^(α) Fmoc, N^(ω) Boc; N^(α) Fmoc, N^(ω) Alloc; N^(α) Boc, N^(ω) Alloc. These combinationsare suitable for peptide synthesis and backbone cyclization, either onsolid support or in solution.

Scheme V Preparation of N^(α), ω-carboxy Protected ω-carboxy AlkyleneAmino Acids.

[0221] One preferred procedure for the preparation of protectedN^(α)(ω-carboxy alkylene) amino acids involves the N^(α)-alkylation ofsuitably N^(α), ω-carboxy deprotected amino acids. One preferreddeprotected amino acid is N^(α)-Benzyl ω-amino acids t-butyl esters(34). This starting material contains one protecting group such as t-Buester which is necessary for the final product, and a temporaryprotecting group such as N^(α) Bzl to minimize side reactions during thepreparation of the titled compound. One preferred procedure for thepreparation of the starting material (34) involves reductive alkylationof ω-amino acids t-butyl esters with aldehydes such as benzaldehyde. Thetemporary protection of the amino group which is used as nucleophile inthe proceeding alkylation reaction by such protecting groups as Bzlminimizes the dialkylation side reaction.

[0222] The N^(α), ω-carboxy deprotected amino acids (34) are reactedwith, for example, chiral α-hydroxy α-substituted acid esters where thehydroxyl moiety is converted to a leaving group, for example, Triflate.The use of Triflate as the leaving group was found to be superior toother leaving groups such as halogens, Tosyl, Mesyl; etc., because itprevents the β-elimination reaction encountered with the other leavinggroups. The use of Triflate as the leaving group also ensures highoptical purity of the product, for example (36). The temporary N^(α)protecting group, such as Bzl, and the α-carboxyl protecting group, suchas benzyl ester, are concomitantly removed by mild condition, such ascatalytic hydrogenation, that to not remove the ω-carboxy protectinggroup such as t-Bu to give the N^(α)(protected ω-carboxy alkylene) aminoacid (36). Introduction of the N^(α) protecting group suitable forpeptide synthesis is accomplished by methods well known in the art, togive the protected N^(α)(ω protected carboxy alkylene) amino acid (37).

[0223] The choice of the N^(α) and the ω-carboxy protecting groups isdictated by the use of the building units in peptide synthesis. Theprotecting groups have to be orthogonal to each other and orthogonal tothe other side chains protecting groups in the peptide. A combination ofN^(α) and ω-carboxy protecting groups are for example: N^(α)-Fmoc,ω-carboxy t-Bu; N^(α)-Fmoc, ω-carboxy Alloc; N^(α)-Boc, ω-carboxy Alloc.These combinations are suitable for peptide synthesis and backbonecyclization, either on solid support or in solution.

Scheme VI Preparation of N^(α), ω-carboxy Protected ω-carboxy AlkyleneGlycine Building Units

[0224] One preferred procedure for the preparation of protectedN^(α)(ω-carboxy alkylene)glycines involves the N^(α)-alkylation ofsuitably N^(α), ω-carboxy deprotected amino acids (34) with commerciallyavailable α-activated carboxylic acid esters for example, benzyl bromoacetate. Since the titled compound is achiral, the use of leaving groupssuch as Trf, Tos or Mes is not necessary.

[0225] The use of the same temporary protecting groups for the N^(α) andthe α-carboxy groups, for example the Bzl protecting group, ensures theprevention of the undesired dialkylation side reaction and allowsconcomitant removal of the temporary protecting groups thus giving highyield of the N^(α)(protected ω-carboxy alkylene)glycines (39).Introduction of the N^(α) protecting group suitable for peptidesynthesis is accomplished by methods well known in the art, to give theprotected N^(α)(ω protected carboxy alkylene)glycines (40).

[0226] The choice of the N^(α) and the ω-carboxy protecting groups isdictated by the use of the building units in peptide synthesis. Theprotecting groups have to be orthogonal to each other and orthogonal tothe other side chains protecting groups in the peptide. A combination ofN^(α) and ω-carboxy protecting groups are, for example: N^(α) Fmoc,ω-carboxy t-Bu; N^(α) Fmoc, ω-carboxy Alloc; N^(α) Boc, ω-carboxy Alloc.These combinations are suitable for peptide synthesis and backbonecyclization, either on solid support or in solution.

Scheme VII Preparation of N^(α) S^(ω)protected ω-thio Alkylene AminoAcid Building Units

[0227] One preferred procedure for the preparation of N^(α),S^(ω)-deprotected N^(α)(ω-thio alkylene) amino acids involves theN^(α)-alkylation of suitably S^(ω) protected ω-thio amino alkanes.Suitable S^(ω) protecting groups are, for example, Bzl, t-Bu, Trt. Onepreferred S^(ω)-protected (ω-thio amino alkanes is for exampleω-(S-Benzyl) amino alkanes (41). One preferred procedure for thepreparation of the starting material (41) involves the use of salts ofS-protected thiols as nucleophiles for a nucleophilic substitutionreaction on suitably N^(α)-protected ω-activated amino alkanes. Removalof the amino protection gives the starting material (41).

[0228] The S-protected ω-thio amino alkanes (41) are reacted with forexample chiral α-hydroxy α-substituted acid esters where the hydroxylmoiety is converted to a leaving group for example Triflate. The use ofTriflate as the leaving group was found to be superior to other leavinggroups such as halogens, Tosyl, Mesyl etc. because it prevents theβ-elimination reaction encountered with the other leaving groups. Theuse of Triflate as the leaving group also ensures high optical purity ofthe product for example (42). The temporary α-carboxyl protecting group,such as methyl ester, is removed by mild condition, such as hydrolysiswith base, that to not remove the ω-thio protecting group such as S-Bzlto give the N^(α) (S-protected ω-thio alkylene) amino acid (43).Introduction of the N^(α) protecting group suitable for peptidesynthesis is accomplished by methods well known in the art, to give theprotected N,S protected N^(α) (ω-thio alkylene) amino acid (44).

[0229] The choice of the N^(α) and the ω-thio protecting groups isdictated by the use of the building units in peptide synthesis. Theprotecting groups have to be orthogonal to each other and orthogonal tothe other side chains protecting groups in the peptide. A combination ofN^(α) and ω-thio protecting groups are for example: N^(α) Fmoc, S^(ω)t-Bu; N^(α) Fmoc, S^(ω) Bzl; N^(α) Fmoc, S^(ω) Trt; N^(α) Boc, S^(ω)Bzl. These combinations are suitable for peptide synthesis and backbonecyclization, either on solid support or in solution.

Scheme VIII Preparation of N^(α), S^(ω) Protected ω-thio AlkyleneGlycine Building Units

[0230] One preferred procedure for the preparation of N^(α),S^(ω)-deprotected N^(α) (ω-thio alkylene) amino acids involves theN^(α)-alkylation of suitably S^(ω) protected ω-thio amino alkanes (41)with commercially available α-activated carboxylic acid esters forexample ethyl bromo acetate. Since the titled compound is achiral, theuse of leaving groups such as Trf, Tos or Mes is not necessary.

[0231] Suitable protecting groups for the ω-thio groups are for exampleBzl, t-Bu, Trt. One preferred S-protected ω-thio amino alkanes is forexample ω-(S-Benzyl) amino alkanes (41). The N-alkylation reaction givesthe ester (45). The temporary α-carboxyl protecting group, such as ethylester, is removed by mild conditions, such as hydrolysis with base, thatto not remove the ω-thio protecting group such as S-Bzl to give theN^(α) (S-protected ω-thio alkylene)glycines (46). Introduction of theN^(α) protecting group suitable for peptide synthesis is accomplished bymethods well known in the art, to give the protected N^(α),S^(ω)-deprotected N^(α) (ω-thio alkylene)glycines (47)

[0232] The choice of the N^(α) and the ω-thio protecting groups isdictated by the use of the building units in peptide synthesis. Theprotecting groups have to be orthogonal to each other and orthogonal tothe other side chains protecting groups in the peptide. A combination ofN^(α) and ω-thio protecting groups are for example: N^(α) Fmoc, S^(ω)t-Bu; N^(α) Fmoc, S^(ω) Bzl; N^(α) Fmoc, S^(ω) Trt; N^(α) Boc, S^(ω)Bzl. These combinations are suitable for peptide synthesis and backbonecyclization, either on solid support or in solution.

Specific Examples of Peptides

[0233] Preparation of the novel backbone cyclized peptide analogs usingthe schematics outlined above will be illustrated by the followingnon-limiting specific examples:

EXAMPLE 1Ada-(D)Arg-Arg-cyclo(N^(α)(1-(6-aminohexylene)Gly-Hyp-Phe-D-Asp)-D-Phe-Phe-Arg-OH

[0234] Stage 1

[0235] Boc-Arg(Tos)-O-resin → Fmoc-Phe-Arg(Tos)-O-resin

[0236] Boc-L-Arg(Tos)-O-resin (0.256 g, 0.1 mmole, 0.39 meq ofnitrogen/g) was placed in a shaker flask and swelled for two hours bythe addition of DCM. The resin was then carried out through theprocedure in Table 1 which includes two deprotections of the Bocprotecting group with 55% TFA in DCM for a total of 22 minutes, washing,neutralization with 10% DIEA in NMP and washing (Table 1 steps 1-8).After positive ninhydrin test, as described in Kaiser et al., AnalBiochem., 34:595, 1970 and is incorporated herein by reference in itsentirety, coupling (Table 1 steps 9-10) was achieved in NMP by theaddition of Fmoc-L-Phe (0.232 g, 0.6 mmole) and after 5 minutes ofshaking, solid BOP reagent (0.265 g, 0.6 mmole) was added to the flask.TABLE 1 PROCEDURE FOR 0.1 mMOLE SCALE STEP SOLVENT/ VOLUME TIME REPEATNO. REAGENT (ML) (MIN) (XS) COMMENT 1 DCM 5 120 1 Swells resin 2 DCM 5 23 3 TFA/DCM 5 2 1 Deprotection 55% 4 TFA/DCM 5 20 1 Deprotection 55% 5DCM 5 2 3 6 NMP 5 2 4 check for positive nin. 7 DIEA/NMP 5 5 2Neutralization 8 NMP 5 2 5 9 Fmoc-AA in 5 5 Coupling add NMP BOP 6 eq.add DIEA 120 600 1 12 eq. Check pH, adjust to pH 8 with DIEA 10 NMP 5 25 check for negative nin. 11 Pip/NMP 5 10 1 Deprotection 20% 12 Pip/NMP5 10 1 20% 13 NMP 5 2 6 check for positive nin.

[0237] After shaking for 10 minutes, the mixture was adjusted to pH 8(measured with wetted pH stick) by the addition of DIEA (0.209 mL, 1.2mmole) and the flask shaken for 10 hours at ambient temperature. Theresin was then washed and subjected to ninhydrin test. After negativeninhydrin test the resin was used for the next coupling.

[0238] Stage 2

[0239] Fmoc-Phe-Arg(Tos)-O-resin → Fmoc-N^(α)(6-Boc aminohexylene)Gly-Hyp(OBzl)-Phe-D-Asp(t-Bu)-D-Phe-Phe-Arg(Tos)-O-resin

[0240] The Fmoc-Phe-Arg(Tos)-O-resin (Stage 1) was subjected to twodeprotections of the Fmoc protecting group by 20% Pip in NMP (Table 1steps 11-13). After washing and ninhydrin test (Method J, below),coupling of Fmoc-D-Phe was achieved as described in Stage 1 (Table 1steps 9-10) using Fmoc-D-phe (0.232 g, 0.6 mmole), BOP reagent (0.265 g,0.6 mmole) and DIEA (0.209 mL, 1.2 mmole). The resin was washed and theFmoc group deprotected as described above (Table 1 steps 11-13). Afterwashing and ninhydrin test, coupling of Fmoc-D-Asp(t-Bu) was achieved asdescribed in Stage 1 (Table 1 steps 9-10) using Fmoc-D-Asp(t-Bu) (0.247g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2mmole). The resin was washed and the Fmoc group deprotected as describedabove (Table 1 steps 11-13). After washing and ninhydrin test, couplingof Fmoc-L-Phe was achieved as described in Stage 1 (Table 1 steps 9-10)using Fmoc-L-Phe (0.232 g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole)and DIEA (0.209 mL, 1.2 mmole). The resin was washed and the Fmoc groupdeprotected as described above (Table 1 steps 11-13). After washing andninhydrin test, coupling of Fmoc-L-Hyp(OBzl) was achieved as describedin Stage 1 (Table 1 steps 9-10) using Fmoc-L-Hyp(OBzl) (0.266 g, 0.6mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2 mmole).The resin was washed and the Fmoc group deprotected as described above(Table 1 steps 11-13). The resin was washed and subjected to picric acidtest (Method K). Coupling of Fmoc-N^(α)(6-Boc amino hexylene)glycine wasachieved as described in Stage 1 (Table 1 steps 9-10) usingFmoc-N^(α)(6-Boc amino hexylene)glycine (0.3 g, 0.6 mmole), BOP reagent(0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2 mmole). The resin was thenwashed and subjected to the picric acid test (Method K below). Afternegative test the resin was used for the next coupling.

[0241] Stage 3

[0242] Fmoc-N^(α)(6-Boc aminohexylene)Gly-Hyp(oBzl)-Phe-D-Asp(t-Bu)-D-Phe-Phe-Arg (Tos)-O-resin →Fmoc-D-Arg(Tos)-Ara(Tos)-N⁶⁰ (6-Boc aminohexylene)Gly-Hyp(OBzl)-Phe-D-Asp(t-Bu)-D-Phe-Phe-Arg(Tos)-O-resin

[0243] The Fmoc-N^(α)(6-Boc aminohexylene)Gly-Hyp(OBzl)-Phe-D-Asp(t-Bu)-D-Phe-Phe-Arg (Tos)-O-resin(Stage 2) was subjected to three deprotection of the Fmoc protectinggroup by 20% Pip in NMP (Table 2 steps 1-2). After washing, the picricacid test (Method K) was performed. If the test did not show 98±2%,deprotection of the peptide resin was subjected again to 3 deprotectionsteps (Table 2 steps 1-2), washing and picric acid test (Method K).Coupling of Fmoc-L-Arg(Tos) was achieved in NMP by the addition of (0.33g, 0.6 mmole) and after 5 minutes of shaking, solid PyBOP reagent (0.28g, 0.6 mmole) was added to the flask. After shaking for 10 minutes, themixture was adjusted to pH 8 (measured with wetted pH stick) by theaddition of DIEA (0.209 mL, 1.2 mmole) and the flask shaken for 2.5hours at ambient temperature. The resin was then washed and subjected toa second coupling by the same procedure for 20 hours. After washing theresin was subjected to picric acid test (Method K) (Table 2 steps 3-6).If the test did not show 98±2% coupling the peptide resin was subjectedagain to a third coupling for 2 hours at 50° C. (Table 2 step 7). Theresin was washed subjected to three deprotection of the Fmoc protectinggroup by 20% Pip in NMP (Table 2 steps 1-2). After washing picric acidtest (Method K) was performed. TABLE 2 PROCEDURE FOR 0.1 mMOLE SCALESTEP SOLVENT/ VOLUME TIME REPEAT NO. REAGENT (ML) (MIN) (XS) COMMENT 1Piperidine/NMP 5 10 3 Deprotection 20% 2 NMP 5 2 6 Picric acid test. 3Fmoc-AA in NMP 5 5 Coupling add PyBroP 6 eq. add DIEA 150 1 12 eq. CheckpH, adjust to pH 8 with DIEA. 4 NMP 5 2 3 check for negative nin. 5Fmoc-AA in NMP 5 5 Coupling add PyBroP 6 eq. add DIEA 20 hr. 1 12 eq.Check pH, adjust to pH 8 with DIEA. 6 NMP 5 2 4 Picric acid test. Ifless than 98 ± 2% coupling repeat Steps 4-5 7 Fmoc-AA in NMP 5 5Coupling at 50° C. add PyBOP 6 eq. add DIEA 120 1 12 eq. Check pH,adjust to pH 8 with DIEA. 8 NMP 5 2 4

[0244] If the test did not show 98±2% deprotection, the peptide resinwas subjected again to 3 deprotection steps (Table 2 steps 1-2), washingand the picric acid test (Method K). Coupling of Fmoc-D-Arg(Tos) wasachieved in NMP as scribed in Stage 1 (Table 1 steps 9-10) usingFmoc-D-Arg(Tos) (0.33 g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole)and DIEA (0.209 mL, 1.2 mmole). The resin was washed 6 times with NMP(Table 1 step 15) and used in the next stages.

[0245] Stage 4

[0246] Fmoc-D-Arg(Tos)-Arg(Tos)-N^(α)(6-Boc aminohexylene)Gly-Hyp(OBzl)-Phe-D-Asp(t-Bu)-D-Phe-Phe-Arg(Tos)-O-resin →Ada-D-Arg-Arg-cyclo(N^(α)(1-(6-amidohexylene)Gly-Hyp-Phe-D-Asp)-D-Phe-Phe-Arg-OH

[0247] The Fmoc-D-Arg(Tos)-Arg(Tos)-N^(α)(6-Boc aminohexylene)Gly-Hyp(OBzl)-Phe-D-Asp(t-Bu)-D-Phe-Phe-Arg(Tos)-O-resin (Stage3) was subjected to deprotection of the Boc and t-Bu protecting groupsand on resin cyclization according to Table 3. The peptide resin waswashed with DCM and deprotected as described in Stage 1 by 55% TFA inDCM. After washing and neutralization by 10% DIEA in NMP and washing 6times with DCM the peptide resin was dried in vacuo for 24 hours. Thedry peptide resin weight, 0.4 g, it was divided into two parts. 0.2 g ofthe peptide resin was swollen 2 hours in 5 mL NMP and cyclized asfollows: Solid TBTU reagent (0.19 g, 6 mmole) was added to the flask.After shaking for 10 minutes, the mixture was adjusted to pH 8 by theaddition of DIEA (0.209 mL, 1.2 mmole) and the flask shaken for 2.5hours at ambient temperature. The resin was then washed and subjected toa second coupling by the same procedure for 20 hours. After washing theresin was subjected to picric acid test (Method K) (Table 3 steps 8-11).If the test did not show 98±2% cyclization the peptide resin wassubjected again to a third cyclization for 2 hours at 50° C. (Table 2step 12). The resin was washed, subjected to three deprotection of theFmoc protecting group by 20% Pip in NMP (Table 2 steps 1-2). Afterwashing and ninhydrin test, the N-terminal amino group was blocked byAda. Adamantane acetic acid (0.108 g, 6 mmole), BOP reagent (0.265 g,0.6 mmole) and DIEA (0.209 mL, 1.2 mmole) were added and the flaskshaken for 2 hours. After washing 6 times with NMP (Table 2 step 13),ninhydrin test (Method J) was performed. If the test was positive orslightly positive the protecting with adamantane acetic acid wasrepeated. If the ninhydrin test was negative, the peptide resin waswashed 6 times with NMP and 6 times with DCM. The resin was dried undervacuum for 24 hours. The dried resin was subjected to HF as follows: tothe dry peptide resin (0.2 g) in the HF reaction flask, anisole (2 mL)was added and the peptide treated with 20 mL liquid HF at −20° C. for 2hours. After the evaporation of the HF under vacuum, the anisole waswashed with ether (20 mL, 5 times) and the solid residue dried invacuum. The peptide was extracted from the resin with TFA (10 mL, 3times) and the TFA evaporated under vacuum. The residue was dissolved in20 mL 30% AcOH and lyophilized. This process was repeated 3 times. Thecrude peptide was purified by semiprep HPLC (Method H). The finalproduct was obtained as white powder by lyophilization from dioxane,which gave 42 mg (56%) of the title compound.

[0248] HPLC (Method G) RT 32.15 minutes, 95%

[0249] TOF MS: 1351.4 (M⁺)

[0250] AAA in agreement with the title compound

[0251] [Table 3 follows at this point.] TABLE 3 PROCEDURE FOR 0.05 mMOLESCALE STEP SOLVENT/ VOLUME TIME REPEAT NO. REAGENT (ML) (MIN) (XS)COMMENT 1 DCM 5 2 3 2 TFA/DCM 55% 5 2 1 Deprotection 3 TFA/DCM 55% 5 201 Deprotection 4 DCM 5 2 3 5 NMP 5 2 4 6 DIEA/NMP 10% 5 5 2Neutralization 7 NMP 5 2 5 8 TBTU/NMP/DIEA 5 150 3 Cyclization 9 NMP 5 24 Picric acid test. If less than 98 ± 2% coupling perform Steps 10-12.If above 98 ± 2%, go to step 13. 10 TBTU/NMP/DIEA 5 20 hr 3 Cyclization.Check pH, adjust to pH 8 with DIEA. 11 NMP 5 2 4 Picric acid test. Ifless than 98 ± 2% coupling perform Steps 12. If above 98 ± 2%, go tostep 13 12 TBTU/NMP/DIEA 5 120 3 Cyclization, 50 C Check pH, adjust topH 8 with DIEA. 13 NMP 5 2 6 14 Pip/NMP 20% 5 10 1 Deprotection 15Pip/NMP 20% 5 10 1 16 NMP 5 2 6 Check for positive nin. 17 AdacOH/BOP/ 52 1 NMP 18 NMP 5 2 6 Check for negative nin. 19 DCM 5 2 4

EXAMPLE 2 Non-cyclized Peptide (Control for Biological Assays)

[0252]Ada-D-Arg-Arg-N^(α)(6-acetamidohexylene)Gly-Hyp-Phe-D-Asp(NH-Me)-D-Phe-Phe-Arg-OH

[0253] The Fmoc-D-Arg(Tos)-Arg(Tos)-N^(α)(6-aminohexylene)Gly-Hyp(OBzl)-Phe-D-Asp-D-Phe-Phe-Arg(Tos)-O-resin (0.2 g)which was prepared in Example 1 Stage 4 was subjected to acetylation ofthe 6-amino side chain of N^(α)(6-acetamidohexylene)Gly and to methylamidation of the carboxylic group of D-Asp as described in Table 4. Thepeptide resin was swollen in 5 mL NMP for 2 hours and AcO (0.113 mL, 12mmole) and PP (17 mg) were added. After 30 minutes, the resin was washedwith NMP 6 times and subjected to ninhydrin test. If the test waspositive or slightly positive the acetylation reaction was repeated. Ifthe ninhydrin test was negative, the carboxy group of D-Asp wasactivated by the addition of HOBT (0.040 g, 0.3 mmole) and DIC (0.047mL, 0.3 mmole) to the peptide resin in NMP. The mixture was shaken forhalf an hour and a solution of 30% methylamine in EtOH (0.2 mL) wasadded. After one hour, the resin was washed 6 times with NMP and theterminal Fmoc group removed by 20% Pip in NMP (Table 4 steps 7-9). Afterwashing with NMP the N-terminal amino group was blocked by Ada asdescribed in Example 1 Stage 4 and the resin was washed with NMP and DCM(Table 4 steps 10-12) and the resin dried in vacuo. The peptide wasdeprotected and cleaved from the resin by HF. To the dry peptide resin(0.2 g) in the HF reaction flask, anisole (2 mL) was added and thepeptide treated with 20 mL liquid HF at −20° C. for 2 hours. After theevaporation of the HF under vacuum, the anisole was washed with ether(20 mL 5 times) and the solid residue dried in vacuo. The peptide wasextracted from the resin with TFA (10 mL, 3 times) and the TFAevaporated under vacuum. The residue was dissolved in 20 mL 30% AcOH andlyophilized. This process was repeated 3 times. The crude peptide waspurified by semiprep HPLC (Method H). The final product was obtained aswhite powder by lyophilization from dioxane, which gave 48 mg (64%) ofthe title compound.

[0254] HPLC (Method G) RT 27.70 minutes, 93%

[0255] TOF MS: 1424.6 (M⁺)

[0256] AAA in agreement with the title compound TABLE 4 PROCEDURE FOR0.05 mMOLE SCALE STEP SOLVENT/ VOLUME TIME REPEAT NO. REAGENT (ML) (MIN)(XS) COMMENT 1 NMP 5 120 1 Swells resin 2 Ac₂O/PP/NMP 5 30 1 Protectingof side chain 3 NMP 5 2 6 Check for negative nin. 4 DIC/HOBT/NMP 5 30 1Activation of COOH side chain 5 MeNH₂/EtOH/ 5 60 1 Protecting of sidechain 6 NMP 5 2 6 7 Pip/NMP 20% 5 10 1 Deprotection 8 Pip/NMP 20% 5 10 19 NMP 5 2 6 Check for positive nin. 10 AdacOH/BOP/ 5 2 1 NMP 11 NMP 5 26 Check for negative nin. 12 DCM 5 2 4

EXAMPLE 3H-D-Arg-Arg-cyclo(N^(α)(1-(4-propanoyl))Gly-Hyp-Phe-N^(α)(3-amido-propylene)Gly)-Ser-D-Phe-Phe-Arq-OH

[0257] Stage 1

[0258] Fmoc-Phe-Arg(Tos)-O-resin →Fmoc-N^(α)(4-t-Bu-propanoyl)Gly-Hyp(OBzl)-Phe-N^(α)(3-Boc aminopropylene)-Gly-Ser(Bzl)-D-Phe-Phe-Arg(Tos)-O-resin

[0259] Fmoc-Phe-Arg(Tos)-O-resin prepared from Boc-Arg(Tos)-O-Resin (0.3g, 0.1 mmole) (Example 1, Stage 1) was subjected to two deprotection ofthe Fmoc protecting group by 20% piperidine in NMP (Table 1, steps11-13). After washing and ninhydrin test (Method J), coupling ofFmoc-D-Phe was achieved as described in Stage 1 (Example 1) (Table 1steps 9-10) using Fmoc-D-Phe (0.232 g, 0.6 mmole), BOP reagent (0.265 g,0.6 mmole) and DIEA (0.209 mL, 1.2 mmole). The resin was washed and theFmoc group deprotected as described above (Table 1, steps 11-13). Afterwashing and ninhydrin test (Method J), coupling of Fmoc-Ser(BzL) wasachieved as described in Stage 1 (Example 1) (Table 1 steps 9-10) usingFmoc-Ser(Bzl) (0.25 g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole) andDIEA (0.209 mL, 1.2 mmole). The resin was washed and the Fmoc groupdeprotected as described above (Table 1, steps 11-13). After washing andpicric acid test (Method K), coupling of Fmoc-N^(α)(3-Boc aminopropylene)glycine was achieved as described in Table 1, steps 9-10 usingFmoc-N^(α)(3-Boc amino propylene)Gly (0.272 g, 0.6 mmole), BOP reagent(0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2 mmole). The resin waswashed and subjected to three deprotection of the Fmoc protecting groupby 20% Pip in NMP (Table 2, steps 1-2). After washing picric acid test(Method K) was performed. If the test did not show 98±2% deprotectionthe peptide resin was subjected again to 3 deprotection steps (Table 2,steps 1-2), washing and picric acid test (Method K). Coupling ofFmoc-L-Hyp(OBzl) was achieved in NMP by the addition of Fmoc-L-Hyp(OBzl)(0.33 g, 0.6 mmole) and after 5 minutes of shaking, solid PyBr0P reagent(0.28 g, 0.6 mmole) was added to the flask. After shaking for 10minutes, the mixture was adjusted to pH 8 by the addition of DIEA (0.209mL, 1.2 mmole) and the flask shaken for 2.5 hours at ambienttemperature. The resin was then washed and subjected to a secondcoupling by the same procedure for 20 hours. After washing the resin wassubjected to picric acid test (Method K) (Table 2, steps 3-6). If thetest did not show 98±2% coupling the peptide resin was subjected againto a third coupling for 2 hours at 50° C. (Table 2, step 7). The resinwas washed subjected to three deprotection of the Fmoc protecting groupby 20% Pip in NMP (Table 2, steps 1-2). After washing picric acid test(Method K) was performed. If the picric acid test did not show 98±2%deprotection, the resin was subjected again to deprotections steps(Table 2, steps 1-2). Coupling of Fmoc-Phe was achieved in NMP by theaddition of Fmoc-Phe (0.232 g, 0.6 mmole), BOP reagent (0.265 g, 0.6mmole) and DIEA (0.209 mL, 1.2 mmole). The resin was washed and afterpicric acid test (Method K) the Fmoc group deprotected as describedabove (Table 2, steps 1-2). After washing picric acid test (Method K)was performed. If the test did not show 98±2% deprotection the peptideresin was subjected again to 3 deprotection steps (Table 2, steps 1-2),washing and picric acid test (Method K). Coupling of N^(α)(3-t-Bucarboxy propylene)Gly was achieved as described in Table 1 steps 9-10using N^(α)(3-t-Bu carboxy propylene)Gly (0.264 g, 0.6 mmole), BOPreagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2 mmole). The resinwas then washed and subjected to the picric acid test (Method K). Afternegative test the resin was used for the next coupling.

[0260] Stage 2

[0261] Fmoc-N^(α)(4-t-Bu-propanoyl)Gly-Hyp(OBzl)-Phe-N^(α)(3-Boc aminopropylene)-Gly-Ser(Bzl)-D-Phe-Phe-Arg(Tos)-O-resin →Fmoc-D-Arg(Tos)-Arg(Tos)-N^(α)(4-t-Bu-propanoyl)Gly-Hyp(OBzl)-Phe-N^(α)(3-Bocamino propylene)-Gly-Ser(Bzl)-D-Phe-Phe-Arg(Tos)-O-resin

[0262] Fmoc-N^(α)(4-t-Bu-propanoyl)Gly-Hyp(OBzl)-Phe-N^(α)(3-Boc aminopropylene)-Gly-Ser(Bzl)-D-Phe-Phe-Arg(Tos)-O-resin (Stage 1) wassubjected to three deprotection of the Fmoc protecting group by 20%Piperidine in NMP (Table 2, steps 1-2). After washing picric acid(Method K) test was performed. If the test did not show 98±2%deprotection the peptide resin was subjected again to 3 deprotectionsteps (Table 6, steps 1-2), washing and picric acid test. Coupling ofFmoc-L-Arg(Tos) was achieved in NMP by the addition of (0.33 g, 0.6mmole) and after 5 minutes of shaking, solid PyBroP reagent (0.28 g, 0.6mmole) was added to the flask. After shaking for 10 minutes, the mixturewas adjusted to pH 8 by the addition of DIEA (0.209 mL, 1.2 mmole) andthe flask shaken for 2.5 hours at ambient temperature. The resin wasthen washed and subjected to a second coupling by the same procedure for20 hours. After washing the resin was subjected to picric acid test(Method K) (Table 2, steps 3-6). If the test did not show 98±2% couplingthe peptide resin was subjected again to a third coupling for 2 hours at50° C. (Table 2, step 7). The resin was washed subjected to threedeprotection of the Fmoc protecting group by 20% Pip in NMP (Table 2,steps 1-2). After washing picric acid test (Method K) was performed. Ifthe test did not show 98±2% deprotection the peptide resin was subjectedagain to 3 deprotection steps (Table 2, steps 1-2), washing and picricacid test (Method K). Coupling of Fmoc-D-Arg(Tos) was achieved in NMP asdescribed in Stage 1 (Table 1, steps 9-10) using Fmoc-D-Arg(Tos) (0.33g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2mmole). The resin was washed 6 times with NMP (Table 1, step 15) andused in the next stages.

[0263] Stage 3

[0264]Fmoc-D-Arg(Tos)-Arg(Tos)-N^(α)(4-t-Bu-propanoyl)Gly-Hyp(OBzl)-Phe-N^(α)(3-Bocamino-propylene)Gly-Ser(Bzl)-D-Phe-Phe-Arc(Tos)-O-resin →H-D-Arg-Arg-cyclo(N^(α)(4-propanoyl))Gly-Hyp-Phe-N^(α)(3-amido-propyl)Gly)-Ser-D-Phe-Phe-Arg-OH

[0265]Fmoc-D-Arg(Tos)-Arg(Tos)-N^(α)(4-t-Bu-propanoyl)Gly-Hyp(OBzl)-Phe-N^(α)(3-Bocamino-propylene)Gly-Ser(Bzl)-D-Phe-Phe-Arg(Tos)-O-resin (Stage 2) wassubjected to deprotection of the Boc and t-Bu protecting groups and onresin cyclization according to Table 5. The peptide resin was washedwith DCM and deprotected as described in Stage 1 by 55% TFA in DCM.After washing and neutralization by 10% DIEA in NMP and washing 6 timeswith and NMP (Table S, steps 1-5) the peptide was cyclized as follow:solid TBTU reagent (0.19 g, 6 mmole) was added to the flask. Aftershaking for 10 minutes, the mixture was adjusted to pH 8 by the additionof DIEA (0.209 mL, 1.2 mmole) and the flask shaken for 2.5 hours atambient temperature. The resin was then washed and subjected to a secondcoupling by the same procedure for 20 hours. After washing the resin wassubjected to picric acid test (Method K) (Table 3, steps 8-11). If thetest did not show 98±2% cyclization the peptide resin was subjectedagain to a third cyclization for 2 hours at 50° C. (Table 2, step 12).The resin was washed, subjected to three deprotection of the Fmocprotecting group by 20% Pip in NMP (Table 5, steps 14-15). After washing6 times with NMP and 4 times with DCM, the resin was dried in vacuo for24 hours. The dried resin was subjected to HF as follows: to the drypeptide resin (0.4 g) in the HF reaction flask, anisole (2 mL) was addedand the peptide treated with 20 mL liquid HF at −20° C. for 2 hours.After the evaporation of the HF under vacuum, the anisole was washedwith ether (20 mL, 5 times) and the solid residue dried in vacuo. Thepeptide was extracted from the resin with TFA (10 mL 3 times) and theTFA evaporated under vacuum. The residue was dissolved in 20 mL 30% AcOHand lyophilized. This process was repeated 3 times. The crude peptidewas purified by semipreparative HPLC (Method H). The final product wasobtained as white powder by lyophilization from dioxane, which gave 59mg (34%) of the title compound.

[0266] [Table 5 follows at this point.] TABLE 5 PROCEDURE FOR 0.1 mMOLESCALE VOL- STEP SOLVENT/ UME TIME REPEAT NO. REAGENT (ML) (MIN) (XS)COMMENT 1 DCM 10 2 3 2 TFA/DCM 10 2 1 Deprotection 55% 3 TFA/DCM 10 20 1Deprotection 55% 4 DCM 10 2 3 5 NMP 10 2 4 6 DIEA/NMP 10 5 2Neutralization 10% 7 NMP 10 2 5 8 TBTU/NMP/ 10 150 3 Cyclization DIEA 9NMP 10 2 4 Picric acid test. If less than 98 ± 2% Coupling perform Steps10. 10 TBTU/NMP/ 10 20 h 3 Cyclization, DIEA Check pH, adjust to pH 8with DIEA. 11 NMP 10 2 4 Picric acid test. If less than 98 ± 2% couplingperform Step 12. 12 TBTU/NMP/ 10 120 3 Cyclization, 50° C. DIEA CheckpH, adjust to pH 8 with DIEA. 13 NMP 10 2 6 14 Piper- 10 10 1Deprotection idine/ NMP 20% 15 Piper- 10 1 idine/ NMP 20% 16 NMP 10 2 6Check for positive nin. 17 DCM 10 2 4

EXAMPLE 4H-D-Arg-Arg-cyclo(N^(α)(4-propanoyl)Gly-Hyp-Phe-N^(α)(3-amido-propyl)-S-Phe)-Ser-D-Phe-Phe-Arg-OH

[0267] Title compound was synthesized according to Example 3 except thatin stage 1, Fmoc-N^(α)(3-Boc-amino-propylene)-S-Phe (0.326) wassubstituted for Fmoc-N^(α)(3-Boc amino propylene)Gly. A total of 0.643 gBoc-L-Arg(Tos)-O-resin (0.39 meq/g, 0.250 mmole) was used and reagentquantities were adjusted accordingly. Cyclic peptide yield (from halfthe total resin used) was 74 mg (42%) of the title compound.

Specific Examples of Building Units

[0268] The following specific examples of novel building units areprovided for illustrative purposes not meant to be limiting. Thefollowing is described in sections, including “Procedures”, “Methods”,“Compounds” and “EXAMPLES”. “Procedures” are detailed stepwisedescriptions of synthetic procedures according to the more generalschemes. “Methods” are general descriptions of analyses used todetermine the progress of the synthetic process. Numbered “Compounds”are either the starting material or intermediates for further numbered“Compounds” the synthesis of which progresses according to the specified“Procedure.” Several “Compounds” used in series produce “EXAMPLES” ofnovel building units of the present invention. For instance, “Compounds”27-29, 41-44, 46-47, 51-55, 62, 63, 67 and 76-79 are actually “EXAMPLES”5-25, respectively, of novel building units of the present invention.“Compounds” 1-26, 30-40, 45, 48-50, 56-61, 64-66, 68-75 are startingmaterials or intermediates (only) for the synthesis of “EXAMPLES”.

[0269] Procedure 1

[0270] Synthesis of N-Boc Alkylene Diamines (BocNH(CH₂)_(n)NH₂) (KnownCompounds).

[0271] To a solution of 0.5 mole alkylene diamine in 0.5 L CHCl₃ cooledin an ice-water bath, was added dropwise, with stirring, a solution of10.91 g (0.05 mole) Boc₂O in 0.25 L CHCl₃ for 3 h. The reaction mixturewas stirred for 16 h. at room temperature and then washed with water(8×250 mL). The organic phase was dried over Na₂SO₄ and evaporated todryness in vacuo.

[0272] Procedure 2

[0273] Synthesis of N-Boc, N-Bzl Alkylene Diamines(BocNH(CH₂)_(n)NH-Bzl).

[0274] To a solution of 0.05 mole of mono Boc alkylene diamine in 60 mLMeOH was added 2.77 mL (0.02 mole) Et₃N, 9.02 g (0.075 mole) MgSO₄, and5.56 mL (0.055 mole) of freshly distilled benzaldehyde. The reactionmixture was stirred under room temperature for 1.5 h. Then 11.34 g (0.3mole) of NaBH₄ were added in small portions during 0.5 h with cooling to−5° C. The reaction mixture was then stirred for 1 h at —5° C. and foranother 1 h at 0° C. The reaction was stopped by addition of 200 mLwater and the product was extracted with EtOAc (3×200 mL). The combinedEtOAc extracts were washed with water (4×100 mL). The organic phase wasextract with 0.5 N HCl (4×100 mL) and the aqueous solution wasneutralized under cooling with 25 mL 25% NH₄OH, extracted with CHCl₃(3×100 ml) and the combined extracts were washed with water (3×80 mL),dried over Na₂SO₄ and evaporated to dryness in vacuo.

[0275] Procedure 3

[0276] Synthesis of (R) or (S) α-hydroxy Acids (Known Compounds).

[0277] To a solution of 16.52 g (0.1 mole) (R) or (S) amino acid in 150ml 1N H₂SO₄ was added dropwise a solution of 10.35 g (0.15 mole) NaNO₂in 100 mL H₂O during 0.5 h with stirring and cooling in an ice bath. Thereaction mixture was stirred 3 h. at 0° C. and additional 18 h at roomtemperature, then the (R)- or (S)- hydroxy acid was extracted with etherin a continuous ether extractor. The etheral solution was washed with 1N HCl (2×50 mL), H₂O (3×80 mL), dried over Na₂SO₄ and evaporated todryness. The product was triturated twice from ether:petrol-ether(40-60° C.) (1:10). The precipitate was filtered, washed with 50 mLpetrol-ether and dried.

[0278] Procedure 4

[0279] Synthesis of (R) or (S) α-hydroxy Acid Methyl Esters (KnownCompounds).

[0280] To a suspension of 0.065 mole (R)- or (S)- hydroxy acid in 100 mLether was added under cooling in an ice bath 300 mL of an etheralsolution of CH₂N₂ until stable yellow color of reaction mixture wasobtained. Then the ether solution was washed with 5% KHCO₃ (3×100 mL)and H₂O (2×80 mL), dried over Na₂SO₄ and evaporated to dryness. Theproduct was dried in vacuo.

[0281] Procedure 5

[0282] Synthesis of Triflate of (R) or (S) α-hydroxy Acid Methyl Esters.

[0283] To a cooled solution of 2.67 ml (0.033 mole) pyridine in 20 mLdry DCM was added 5.55 mL (0.033 mole) Trf₂O at −20° C. (dry ice in EtOHbath), then after 5 min a solution of 0.03 mole (R) or (S) α-hydroxyacid methyl ester in 20 mL dry DCM was added dropwise. The reactionmixture was stirred at room temperature for 45 min, then was passedthrough a short silica gel column (2 cm). The product was eluted with400 mL of petrol-ether:methylene-chloride (1:1). The solvent wasevaporated in vacuo.

[0284] Procedure 6

[0285] Synthesis of (R) or (S) N^(α) (Bzl)(N^(ω)-Boc-aminoAlkylene)Amino Acid Methyl Esters ((R) or (S)BocNH(CH₂)_(n)N(Bzl)CH(R)COOMe).

[0286] To a solution of 0.022 mole of N^(α)-Boc, N^(ω)-Bzl alkylenediamine in 20 mL of dry DCM was added 3.04 mL (0.022 mole) Et₃N. Then asolution of 0.02 mole of (R) or (S) α-hydroxy acid methyl ester triflatein 25 mL dry DCM was added dropwise (0.5 h.) under cooling in anice-water bath. The reaction mixture was stirred at room temperature for18 h. Then 150 mL of CHCl₃ was added and the yellow solution was washedwith water (3×80 mL). The organic phase was dried over Na₂SO₄ andadsorbed on silica-gel and dried in vacuo. The silica-gel was washed onfilter with 0.5 L of petrol-ether and with 0.5 L of 2% EA in PE. Thenthe product was eluted from silica with 0.5 L of mixturepetrol-ether:ethyl-acetate (4:1). The solvent was evaporated in vacuo.If the product was not clean it was further purified on a small columnof silica-gel (250 mL). The first impurities were eluted with 0.8 L ofhexane then the product was eluted with 1.5 L of mixture ofpetrol-ether: ethyl-acetate (4:1).

[0287] Procedure 7

[0288] Hydrolysis of Methyl Esters

[0289] To a solution of 0.015 mole of methyl ester in 40 mL MeOH wasadded 10 mL 7.5 N NaOH cooled in an ice-water bath. The reaction mixturewas stirred at room temperature for approximately 24 h (until the methylester spot disappears on TLC). Then 100 mL of water were added and thereaction mixture was washed with petrol-ether (3×80 mL). The aqueoussolution was acidified under cooling by addition of 40 mL 2N HCL. Theproduct was extracted with a mixture of CHCl₃:i-PrOH (3:1) (3×80 mL),dried over Na₂SO₄, evaporated to dryness and dried in vacuo to obtain awhite foam in quantitative yield.

[0290] Procedure 8

[0291] Removal of Bzl by Hydrogenation with Pd/C

[0292] To a solution of 0.012 mole (R) or (S) N^(α)(Bzl)(N^(ω)-Boc-amino alkylene)amino acid in 60 mL MeOH-DMF (11-1) wasadded 0.5 g 10% Pd/C. The solution was hydrogenated for 4 h under apressure of 45-50 Psi at room temperature. Then 200 mL of a mixture ofDMF:MeOH:H₂O:glacial AcOH (1:3:5:1) was added. The catalyst wasfiltrated off and washed (is the acetic acid?) with H₂O or MeOH (2×15mL). The combined filtrate was evaporated to dryness and recrystallizedfrom methanol: ether (15 mL:250). The precipitate was filtered and driedin vacuo.

[0293] Procedure 9

[0294] Synthesis of (R) or (S) N^(α) (Fmoc)(N^(ω)-Boc-amino alkylene)Amino Acid.

[0295] To 50 mL water was added 0.07 mole of (R) or (S) N^(α)(N^(ω)-Boc-amino alkylene) amino acid and 1.95 mL (0.014 mole) Et₃N. Thesuspension was stirred 2-3 h until a clear solution was obtained. Then asolution of 2.25 g (0.07 mole) of FmocOSu in 100 mL ACN was added. Thereaction mixture was stirred 18 h at room temperature, then 150 mL waterwas added and the solution was washed with petrol-ether (3×100 mL) andwith ether:petrol-ether (1:4). The aqueous solution was acidified byaddition of 14 mL 1N HCL. The product was extracted with EtOAc (4×100mL) and the organic phase was washed with 0.5 N HCl (2×50 mL), H₂O (3×80mL), dried over Na₂SO₄, evaporated to dryness and recrystallized fromether: petrol-ether (80 mL: 200 mL)

[0296] Procedure 10

[0297] Synthesis of S-benzylcysteamine (Bzl-S—(CH₂)₂—NH₂) (KnownCompound).

[0298] To a suspension of 0.1 mole cysteamine hydrochloride in 20 mLmethanol were added 13.6 mL of 25% ammonia solution, followed bydropwise addition of 0.12 mole benzyl bromide at room temperature. Themixture was stirred for 0.5 h, and the formed precipitate ofS-dibenzylcysteamine was collected by filtration. The product wasextracted with ether (3×100 mL) and the organic phase was successivelywashed with brine (2×100 mL), dried over MgSO₄ and the solventevaporated in vacuo. The crude product was essentially pure enough forthe next step. It could, however be recrystallized from ethyl acetate.Yield 86%, of white solid. m.p. 85-6° C. NMR (CDCl₃) in agreement withthe title compound.

[0299] Procedure 11

[0300] Synthesis of N^(α)-(ω-(benzylthio)alkylene)phthalimides((Bzl-S—(CH₂)_(n)—N=Pht)) (Known Compounds).

[0301] N-(ω-bromoalkylene)phthalimide (0.1 mole) and benzyl mercaptane(0.11 mole) were stirred with 0.1 mole potassium carbonate in 100 mLDMSO at 50° C. for 24 hours. The mixture was poured into ice-water andthe product was allowed to crystallize for 0.5 hour, collected byfiltration and recrystallized from i-PrOH.

[0302] Procedure 12

[0303] Synthesis of N^(α)-(ω-(benzylthio)alkyl) Amines(Bzl-S—(CH₂)_(n)—NH₂) (Known Compounds).

[0304] Hydrazinolysis of the phthalimide group was performed byrefluxing 0.09 mole of N^(α)-(ω-(benzylthio)alkylene)-phthalimide(Procedure 11) with 120 mL 1M solution of hydrazine hydrate in ethanol(diluted with additional 220 mL ethanol) for 2 hours. The formedprecipitate was collected by filtration and hydrolyzed with 180 mL 2NHCl at 50° C. for 0.5 hour. The water was evaporated in vacuo and thecrude hydrochloride dissolved in 50 mL 25% ammonia solution. The freeamine was extracted with DCM (4×100 mL) and the organic phase was washedwith brine (2×100 mL), dried over MgSO₄ and the solvent evaporated invacuo. The crude N-(ω-(benzyl-thio)alkyl)amine was distilled underreduced pressure, and appeared as colorless oil, which could be keptrefrigerated under nitrogen for prolonged periods.

[0305] Procedure 13

[0306] Synthesis of (R) and (S) N^(α)-(ω-(benzylthio)alkylene) AminoAcids Methyl Esters ((R) or (S)(Bzl-S—(CH₂)_(n)—NH—CH(R)—COOMe)).

[0307] To a solution of 15 mmol N-(ω-(benzylthio)alkyl)amine and 15 mmolDIEA in 55 mL DCM was added dropwise a solution of 15 mmol (R) or (S)α-hydroxy acid methyl ester triflate (Procedure 5) in 55 mL DCM at 0° C.The reaction mixture was then stirred at room temperature for 18 h. Themixture was then diluted with 100 mL DCM and washed with water (3×100mL). The crude product was cleaned on a silica-gel column withDCM:MeOH(99:1), and was further crystallized from DIE:hexane.

[0308] In the case of Glycine, bromoacetic acid esters were suitablestarting materials. Identical results were obtained when both thesesubstrates were reacted with N-(ω-(benzylthio) alkyl)amines.

[0309] Procedure 14

[0310] Synthesis of (R) or (S) Boc-N^(α)-(ω-(benzylthio)alkylene)AminoAcids ((R) or (S)(Bzl-S—(CH₂)_(n)—N(Boc)—CH(R)—COOH))

[0311] 10 mmol (R) or (S) N-(ω-(benzylthio)alkylene) amino acid methylester was dissolved in 50 mL 1,4-dioxane and 50 mL 1N NaOH were added.The mixture was stirred at room temperature overnight. The disappearanceof the starting material was followed by TLC (Silicagel+F₂₅₄,CHCl₃:MeOH-1:4). When all the ester was hydrolyzed, 50 mL waterwere added, followed by 30 mmol Boc₂O. The mixture was stirredovernight, then the dioxane was evaporated in vacuo, the mixture wascooled in an ice-water bath, covered with 100 mL EtAc and acidified withsaturated KHSO₄ to pH 2-3. The layers were separated, and the aqueouslayer was extracted with additional 2×100 mL EtOAc. The organic layerwas washed with water (2×100 mL), dried over MgSO₄ and the solventevaporated in vacuo. The crude product was cleaned on a silica-gelcolumn with DCM:MeOH-99:1 or crystallized from DIE:hexane.

[0312] Procedure 15

[0313] Synthesis of Dipeptides (S,S)-Boc-aminoAcid-N^(α)-(ω-(benzylthio)alkylene) Amino Acids Esters.

[0314] A solution of 1.1 mmol Boc-amino acid, 1 mmolN^(α)-(ω-(benzylthio)alkylene) amino acid ester, 1.1 mmol BOP and 3 mmolDIEA in 10 mL DCM was stirred at room temperature for 2 hours. Then themixture was diluted with 40 mL of DCM and washed successively withsaturated KHSO₄ (3×100 mL), saturated KHCO₃ (3×100 mL) and brine (2×100mL), dried over MgSO₄ and the solvent evaporated in vacuo. The crudeproduct was cleaned on a silica-gel column with DCM:MeOH-99:1 orcrystallized from DIE:hexane.

[0315] Procedure 16

[0316] Synthesis of N^(α)-Bzl ω-amino Acids t-butyl Esters(Bzl-NH—(CH₂)_(n)—COO-t-Bu).

[0317] A solution of 0.05 mole of amino acid t-butyl ester acetate in200 mL H₂O was acidified to pH 2 with AcOH, washed with PE (70 mL X5)cooled and the pH adjusted to 9 by NH₄OH 25%. The free amino acidt-butyl ester was extracted with i-Pr:CHCl (3:1, 3×100 mL). The combinedextracts were dried on Na₂SO₄ and evaporated to dryness under vacuum.The benzylation reaction was performed according to Procedure 2.

[0318] Procedure 17

[0319] Synthesis of N^(α)-(Bzl) (ωt-Bu Carboxy Alkylene) Glycine BzlEster (N-(Bzl)(CH₂)_(n)—COO-t-Bu) CH₂—COO-Bzl)

[0320] To a stirred solution of 0.015 mole of N-Bzl ω-amino acid t-butylester in 10 mL DMF at 0° C. were added 2.61 mL of DIEA and 2.38 mL ofbenzyl bromo acetate. The reaction mixture was stirred 30 min at 0° C.and 3 h at room temperature. After the addition of 200 mL of ether, theprecipitate was 25 removed by filtration and the organic phase washedwith H₂O (3×80 mL), 1N HCl (3×80 mL), H₂O (3×80 mL), dryed over Na₂SO₄and evaporated to dryness under vacuum. The resulting oil was driedunder vacuum.

[0321] Methods

[0322] ANALYTICAL TLC was performed on TLC plates of silica gel E (MerckF₂₅₄), using the following solvent systems METHOD A DCM:MeOH:AcOH16:4:0.5 METHOD B PE:EtOAc  1:1 METHOD C PE:EtOAc  4:1 METHOD DCHCl₃:EtOAc  4:1 METHOD E PE:EtOAC  9:1 METHOD F CHCl₃:EtOAc 19:1

[0323] METHOD G ANALYTICAL REVERSE PHASE HPLC Column Merck LICHROCARTRP-18 5 μm, 250 × 4 mm. Mobile Phases A = 0.1% TFA in H₂O B = 0.1% TFAin ACN Gradient T = 0-5 min A(75%), B(25%) T = 30 min A(50%), B(50%) T =40 min A(100%), B(0%) T = 50 min A(100%), B(0%)

[0324] METHOD H—SEMIPREPARATIVE REVERSE PHASE HPLC The crude peptide wasdissolved in MeOH (1 mL) and chromatographed using reverse phasesemipreparative HPLC with the following conditions: Column Merck HibarLICHROSORB RP-18 7 μm, 250 × 410 mm Mobile Phases A = 0.1% TFA in H₂O B= 0.1% TFA in ACN Gradient T = 0-10 min A(80%), B(20%) T = 60 minA(100%) , B(0%) T = 70 min A(100%), B(0%)

[0325] METHOD J NINHYDRIN TEST (NIN. TEST)

[0326] The test was performed according to Kaiser et al. Anal. Biochem.,34:595 (1970) which is incorporated herein by reference in its entirety.Test was considered negative when the resin did not change color afterheating to 11° C. for 2 minutes with the test mixture. Test wasconsidered positive or slightly positive when the resin was dark orfaint purple after heating to 110° C. for 2 minutes with the testmixture.

[0327] Method K—Quantitative Picric Acid Test

[0328] Picric acid test was performed after removal of the Fmocprotecting group from the amino acid preceding the coupling of protectedNα(ω-alkylene) building unit. This absorbance was taken as 100% freeamines. After coupling the test was used to check yield of % coupling bycomparison.

[0329] The resin 0.1 mmole was treated according to steps 1-8, Table 1.After step 8 the resin was introduced into a centrifuge tube and shakenwith 40 mL of 5% DIEA:95% DCM for ten minutes. The resin was centrifuged5 minutes and 4 mL of the solution were pipette into 40 mL. EtOH and theabsorbance at 358 nm measured. This procedure was repeated 3 times andthe average absorbance calculated. TABLE 6 PROCEDURE FOR 0.1 mMOLE SCALESTEP SOLVENT/ VOLUME TIME REPEAT COM- NO. REAGENT (ML) (MIN) (XS) MENT 1NMP 10 1 3 2 DCM 10 1 2 3 Picric acid 10 1 2 0.05 M/DCM 4 DMF 10 0.5 105 NMP 10 1 2 6 NMP 10 2 3 7 10% EtOH/DCM 10 1 1 8 DCM 10 1 2

[0330] Compound 1

[0331] N-Boc Diamino Ethane (Known Compound)

[0332] A solution of 33.4 mL of ethylene diamine in CHCl₃ and 10.91 g ofBoc₂O were used (Procedure 1). Yield 97% of colorless oil.

[0333] TLC (Method A) Rf 0.2-0.24 (one spot)

[0334] NMR (CDCl₃) in agreement with the title compound

[0335] Compound 2

[0336] N-Boc 1,3 Diamino Propane (Known Compound).

[0337] A solution of 41.7 mL of 1,3 diamino propane in CHCl₃ and 10.91 gof Boc₂O were used (Procedure 1). Yield 96% of colorless oil.

[0338] TLC (Method A) Rf 0.27-0.3 (one spot)

[0339] NMR (CDCl₃) in agreement with the title compound

[0340] Compound 3

[0341] N-Boc 1,4 diamino butane (Known Compound).

[0342] A solution of 44.08 g of 1,4 diamino butane and 10.91 g of Boc₂Owere used (Procedure 1). Yield 98% of white oil.

[0343] TLC (Method A) Rf 0.32-0.35 (one spot).

[0344] NMR (CDCl₃) in agreement with the title compound.

[0345] Compound 4

[0346] N-Boc 1,6 diamino hexane (Known Compound).

[0347] A solution of 58.10 g of 1,6 diamino hexane in CHCl₃ and 10.91 gof Boc₂O were used (Procedure 1). Yield 70% of colorless oil afterpurification on a silica gel column and elution with CHCl₃-MeOH (4:1).

[0348] TLC (Method A) Rf 0.50-0.54 (one spot)

[0349] NMR (CDCl₃) in agreement with the title compound

[0350] Compound 5

[0351] N-Boc, N-Bzl 1,2 diamino ethane

[0352] A solution of 8.01 g of Boc ethylene diamine (COMPOUND 1) wasused (Procedure 2). Yield 65% of colorless oil.

[0353] TLC (Method A) Rf 0.62-0.65 (one spot)

[0354] NMR (CDCl₃) in agreement with the title compound

[0355] Compound 6

[0356] N-Boc, N-Bzl, 1,3 diamino propane.

[0357] A solution of 8.71 g of N-Boc 1,3 diamino propane (COMPOUND 2)was used (Procedure 1). Yield 75% of colorless.

[0358] TLC (Method A) Rf 0.63-0.68 (one spot)

[0359] NMR (CDCl₃) in agreement with the title compound

[0360] Compound 7

[0361] N-Boc, N-Bzl 1,4 diamino butane

[0362] A solution of 9.41 g of N-Boc 1,4 diamino butane (Compound 3) wasused (Procedure 1). Yield 63% of white oil.

[0363] TLC (Method A) Rf 0.65-0.72 (one spot)

[0364] NMR (CDCl₃) in agreement with the title compound

[0365] Compound 8

[0366] N-Boc, N-Bzl 1,6 diamino hexane

[0367] A solution of 10.82 g of N-Boc 1,6 diamino hexane (Compound 4)was used (Procedure 1). The ethyl acetate solution after extraction wasdried over Na₂SO₄ and evaporated under vacuum to dryness. The remainingcrude product was dissolved in 400 mL chloroform and washed with 0.5 NHCl (3×80 mL, 0.12 mole), water (2×100 mL) dried over Na₂SO₄ andevaporated to dryness. Then 200 mL of ether was added. The precipitatewas filtered, washed with ether (3×50 mL) and dried under vacuum.

[0368] Yield 70% of white solid mp 150-152° C.

[0369] TLC (Method A) Rf 0.8 (one spot)

[0370] To remove HCl from product with n=6, the HCl salt was dissolvedin CHCl₃, washed with an alkali solution (0.5% NH₄OH), dried over Na₂SO₄and evaporated to dryness. NMR (CDCl₃) in agreement with the titlecompound.

[0371] Compound 9

[0372] (S)-3-Phenylacetic acid.methyl ester (Known Compound)

[0373] A suspension of 10.8 g of (S)-3-Phenylacetic acid in 100 mL etherwas treated with diazomethane (Procedure 4).

[0374] Yield 85.

[0375] TLC (Method B) Rf 0.6-0.65 (one spot)

[0376] (α)_(D)=+3,3 (c=1, MeOH)

[0377] NMR (CDCl₃) in agreement with the title compound

[0378] Compound 10

[0379] (R)-3-Phenylacetic acid methyl ester (Known Compound).

[0380] A suspension of 10.8 g (R)-3-Phenylacetic acid in 100 mL etherwas treated with diazomethane (Procedure 4). Yield 84%.

[0381] TLC (Method B) Rf 0.6-0.65 (one spot)

[0382] (α)_(D)=−3,3 (c=1, MeOH)

[0383] NMR (CDCl₃) in agreement with the title compound.

[0384] Compound 11

[0385] (S)—O-Trf-3-Phenylacetic acid methyl ester

[0386] To a cooled solution of Trf₂O and pyridine in dry DCM (Procedure5), a solution of 5.4 g of (S)-3-Phenylacetic acid methyl ester wasadded. After the workup (Procedure 5), the yield was 74%. The productwas used immediately or kept in a cold desiccator under Ar.

[0387] Compound 12

[0388] (R)—O-Trf-3-Phenylacetic acid methyl ester

[0389] To a cooled solution of Trf₂O and pyridine in dry DCM (Procedure5), a solution of 5.4 g of (R)-3-Phenylacetic acid methyl ester wasadded. After the workup (Procedure 5), the yield was 74%. The productwas used immediately or kept in a cold desiccator under Ar.

[0390] Compound 13

[0391] N^(α) (Bzl)(2-Boc-amino ethylene) (R)Phenylalanine methyl ester

[0392] A solution of 6.24 g of (S)—O-Trf-3-Phenyllactic acid methylester in dry DCM (Compound 11) was added to a solution of 5.51 g ofN-Boc, N-Bzl-diamino ethane (Compound 5) in dry DCM (Procedure 6). Yield69.2%

[0393] (α)_(D)=+64.0 (c=1, MeOH)

[0394] TLC (Method C) Rf=0.41 (one spot)

[0395] NMR (CDCl₃) in agreement with the title compound.

[0396] Compound 14

[0397] N^(α) (Bzl)(3-Boc-amino propylene) (S)Phenylalanine methyl ester

[0398] A solution of 6.24 g of (R)—O-Trf-3-Phenyllactic acid methylester in dry DCM (Compound 12) was added to a solution of 5.82 g ofN-Boc, N-Bzl-diamino propane (Compound 6) in dry DCM (Procedure 6).Yield 67.7%

[0399] (α)_(D)=−55.8 (c=1, MeOH)

[0400] TLC (Method C) Rf=0.38 (one spot)

[0401] NMR (CDCl₃) in agreement with the title compound.

[0402] Compound 15

[0403] N^(α) (Bzl)(4-Boc-amino butylene) (S)Phenylalanine methyl ester

[0404] A solution of 6.24 g of O-Trf-(R)-3-Phenyllactic acid methylester in dry DCM (Compound 12) was add to a solution of 6.12 g of N-Boc,N-Bzl-diamino butane (Compound 7) in dry DCM (Procedure 6). Yield 58.6%

[0405] (α)_(D)=−62.6 (c=1, MeOH)

[0406] Rf (Method C ) 0.42 (one spot)

[0407] NMR (CDCl₃) in agreement with the title compound.

[0408] Compound 16

[0409] N^(α) (Bzl)(6-Boc-amino hexylene) (S)Phenylalanine methyl ester

[0410] A solution of 6.24 g of O-Trf-(R)-3-Phenyllactic acid methylester in dry DCM (Compound 12) was add to a solution of 6.74 g of N-Boc,N-Bzl-diamino hexane (Compound 8) in dry DCM (Procedure 6). Yield 78.9%

[0411] (α)_(D)=−60.0 (c=1, MeOH)

[0412] TLC (Method C) Rf=0.47 (one spot)

[0413] NMR (CDCl₃) in agreement with the title compound.

[0414] Compound 17

[0415] N^(α) (Bzl)(3-Boc-amino propylene) (R)Phenylalanine methyl ester

[0416] A solution of 6.24 g of O-Trf-(S)-3-Phenyllactic acid methylester in dry DCM (Compound 11) was add to a solution of 5.82 g of N-Boc,N-Bzl-diamino propane (Compound 6) in dry DCM (Procedure 6). Yield 51.5%

[0417] (α)_(D)=+58.8 (c=1, MeOH)

[0418] TLC (Method C) Rf=0.35 (one spot)

[0419] NMR (CDCl₃) in agreement with the title compound.

[0420] Compound 18

[0421] N^(α) (Bzl)(4-Boc-amino butylene) (R)Phenylalanine methyl ester

[0422] A solution of 6.24 g of O-Trf-(S)-3-Phenyllactic acid methylester in dry DCM (Compound 11) was add to a solution of 6.12 g of N-Boc,N-Bzl-diamino butane (Compound 7) in dry DCM (Procedure 6). Yield 66.8%

[0423] (α)_(D)=+59.0 (c=1, MeOH)

[0424] TLC (Method C) Rf=0.33 (one spot)

[0425] NMR (CDCl₃) in agreement with the title compound.

[0426] Compound 19

[0427] N^(α) (Bzl)(3-Boc-amino propylene) (S)Phenylalanine

[0428] A solution of 6.61 g of N^(α) (Bzl)(3-Boc-amino propylene)(S)Phenylalanine methyl ester in MeOH (Compound 14) was hydrolyzed byNaOH 7.5N (Procedure 7). Yield 89.5%

[0429] (α)_(D)=−24.0 (c=1, MeOH)

[0430] TLC (Method B) Rf=0.16 (one spot)

[0431] NMR (CDCl₃) in agreement with the title compound.

[0432] Compound 20

[0433] N^(α) (Bzl)(4-Boc-amino butylene) (S)Phenylalanine

[0434] A solution of 6.61 g of N^(α) (Bzl)(4-Boc-amino butylene)(S)Phenylalanine methyl ester in MeOH (Compound 15) was hydrolyzed byNaOH 7.5N (Procedure 7). Yield 73.5%

[0435] (α)_(D)=−12.0 (c=1, MeOH)

[0436] TLC (Method D) Rf=0.6 (one spot)

[0437] NMR (CDCl₃) in agreement with the title compound.

[0438] Compound 21

[0439] N^(α) (Bzl)(3-Boc-amino propylene) (R)Phenylalanine

[0440] A solution of 6.40 g of N^(α) (Bzl)(3-Boc-amino propylene)(R)Phenylalanine methyl ester in MeOH (Compound 17) was hydrolyzed byNaOH 7.5N (Procedure 7). Yield 100%

[0441] (α)_(D)=+15.33 (c=1, MeOH)

[0442] TLC (Method B) Rf=0.38 (one spot)

[0443] NMR (CDCl₃) in agreement with the title compound.

[0444] Compound 22

[0445] N^(α) (Bzl)(4-Boc-amino butylene) (R)Phenylalanine

[0446] A solution of 6.61 g of N^(α) (Bzl)(4-Boc-amino butylene)(R)Phenylalanine methyl ester in MeOH (Compound 18) was hydrolyzed byNaOH 7.5N (Procedure 7). Yield 100%

[0447] (α)_(D)=+12.0 (c=1, MeOH)

[0448] TLC (Method D) Rf=0.54 (one spot)

[0449] NMR (CDCl₃) in agreement with the title compound.

[0450] Compound 23

[0451] N^(α) (3-Boc-amino propylene) (S)Phenylalanine HCl

[0452] A solution of 5.39 g of N^(α) (Bzl)(3-Boc-amino propylene)(S)Phenylalanine (Compound 19) in MeOH-DMF was hydrogenated on Pd/C(Procedure 8). Yield 86.1%

[0453] TLC (Method A)Rf=0.51 (one spot)

[0454] NMR (D₂O+Na₂CO₃) in agreement with the title compound.

[0455] Compound 24

[0456] N^(α) (4-Boc-amino butylene) (S)Phenylalanine HCl

[0457] A solution of 5.56 g of N^(α) (Bzl)(4-Boc-amino butylene)(S)Phenylalanine (Compound 20) in MeOH-DMF was hydrogenated on Pd/C(Procedure 8). Yield 79.25%

[0458] TLC (Method A) Rf=0.50 (one spot)

[0459] NMR (D₂O+Na₂CO₃) in agreement with the title compound.

[0460] Compound 25

[0461] N^(α) (3-Boc-amino propylene) (R)Phenylalanine HCl

[0462] A solution of 5.39 g of N^(α) (Bzl)(3-Boc-amino propylene)(R)Phenylalanine (Compound 21) in MeOH-DMF was hydrogenated on Pd/C(Procedure 8). Yield 75.5%

[0463] TLC (Method A) Rf=0.51 (one spot)

[0464] NMR (D₂O+Na₂CO₃) in agreement with the title compound.

[0465] Compound 26

[0466] N^(α) (4-Boc-amino butylene) (R)Phenylalanine HCl

[0467] A solution of 5.56 g of N^(α) (Bzl)(4-Boc-amino butylene)(R)Phenylalanine (Compound 22) in MeOH-DMF was hydrogenated on Pd/C(Procedure 8). Yield 73.85%

[0468] TLC (Method A) Rf=0.50 (one spot)

[0469] NMR (D₂O+Na₂CO₃) in agreement with the title compound.

EXAMPLE 5

[0470] Compound 27

[0471] N^(α) (Fmoc)(3-Boc-amino Propylene)(S)Phenylalanine

[0472] A solution of 2.51 g of N^(α) (3-Boc-amino propylene)(S)Phenylalanine.HCl (Compound 23) in H₂O-ACN was reacted with FmocOSu(Procedure 9). Yield 64.84%

[0473] TLC (Method D) Rf=0.74 (one spot)

[0474] (α)_(D)=−87 (c=1, MeOH)

[0475] HPLC (Method G) 92%

[0476] NMR (CDCl₃) in agreement with the title compound.

EXAMPLE 6

[0477] Compound 28

[0478] N^(α) (Fmoc)(3-Boc-amino propylene) (R)Phenylalanine

[0479] A solution of 2.51 g of N^(α) (3-Boc-amino propylene)(R)Phenylalanine.HCl (Compound 25) in H₂O-ACN was reacted with FmocOSu(Procedure 9). Yield 61.56%

[0480] TLC (Method D) Rf=0.62 (one spot)

[0481] (α)_(D)=+79.6 (c=1, MeOH)

[0482] HPLC (Method G) 94%

[0483] NMR (CDCl₃) in agreement with the title compound.

EXAMPLE 7

[0484] Compound 29

[0485] N^(α) (Fmoc)(4-Boc-amino butylene) (S)Phenylalanine

[0486] A solution of 2.61 g of N^(α) (4-Boc-amino butylene)(S)Phenylalanine.HCl (Compound 24) in H₂O-ACN was reacted with FmocOSu(Procedure 9). Yield 56%

[0487] TLC (Method D) Rf=0.64 (one spot)

[0488] HPLC (Method G) 89%

[0489] NMR (CDCl₃) in agreement with the title compound.

[0490] Compound 30

[0491] O-Trf-(S)-Lactic acid methyl ester

[0492] To a cooled solution of Trf₂O and pyridine in DCM (Procedure 5),a solution of 2.9 mL of (S)lactic acid methyl ester was added. After theworkup (Procedure 5), the yield was 70%. The product was usedimmediately or kept in a cold desiccator under Ar.

[0493] Compound 31

[0494] O-Trf-(R)-Lactic acid methyl ester

[0495] To a cooled solution of Trf₂O and pyridine in DCM (Procedure 5),a solution of 2.9 mL of (R) lactic acid methyl ester was added. Afterthe workup (Procedure 5), the yield was 70%. The product was usedimmediately or kept in a cold desiccator under Ar.

[0496] Compound 32

[0497] N^(α) (Bzl)(3-Boc-amino propylene) (S)Alanine methyl ester

[0498] A solution of 4.72 g of O-Trf-(R)-lactic acid methyl ester in dryDCM (Compound 31) was add to a solution of 5.82 g of N-Doc,N-Bzl-diamino propane (Compound 6) in dry DCM (Procedure 6). Yield 69.5%

[0499] (α)_(D)=−6.6 (C=1, MeOH)

[0500] TLC (Method C) Rf=0.42 (one spot)

[0501] TLC (Method D) Rf=0.92 (one spot)

[0502] TLC (Method E) Rf=0.13 (one spot)

[0503] NMR (CDCl₃) in agreement with the title compound.

[0504] Compound 33

[0505] N^(α) (Bzl)(3-Boc-amino propylene) (R)Alanine methyl ester

[0506] A solution of 4.72 g of O-Trf-(S-lactic acid methyl ester in dryDCM (Compound 30) was add to a solution of 5.82 g of N-Doc,N-Bzl-diamino propane (Compound 6) in dry DCM (Procedure 6). Yield 71%

[0507] (α)_(D)=+6.53 (C=1, MeOH)

[0508] TLC (Method C) Rf=0.42 (one spot)

[0509] TLC (Method D) Rf=0.93 (one spot)

[0510] NMR (CDCl₃) in agreement with the title compound.

[0511] Compound 34

[0512] N^(α) (Bzl)(6-Boc-amino hexylene) (S)Alanine methyl ester

[0513] A solution of 4.72 g of O-Trf-(R)-lactic acid methyl ester in dryDCM (Compound 31) was add to a solution of 6.74 g of N-Boc,N-Bzl-diamino hexane (Compound 8) in dry DCM (Procedure 6). Yield 81.42%

[0514] (α)_(D)=−6.76 (C=1, MeOH)

[0515] TLC (Method D) Rf=0.95 (one spot)

[0516] TLC (Method E) Rf=0.26 (one spot)

[0517] NMR (CDCl₃) in agreement with the title compound.

[0518] Compound 35

[0519] N^(α) (Bzl)(2-Boc-amino propylene) (S)Alanine

[0520] A solution of 5.25 g of N^(α)(Bzl)(2-Boc-amino ethylene)(S)Alanine methyl ester in MeOH (Compound 32) was hydrolyzed by NaOH7.5N (Procedure 7). Yield 100% of white solid, mp 64° C.

[0521] (α)_(D)=+0.5 (C=1, MeOH)

[0522] TLC (Method A) Rf=0.64 (one spot)

[0523] TLC (Method D) Rf=0.47 (one spot)

[0524] NMR (CDCl₃) in agreement with the title compound.

[0525] Compound 36

[0526] N^(α) (Bzl)(6-Boc-amino hexylene) (S)Alanine

[0527] A solution of 5.88 g of N^(α) (Bzl)(6-Boc-amino hexylene)(S)Alanine methyl ester (Compound 34) in MeOH was hydrolyzed by NaOH7.5N (Procedure 7). Yield 100%

[0528] (α)_(D)=+0.7 (C=1, MeOH)

[0529] TLC (Method D) Rf=0.51 (one spot)

[0530] NMR (CDCl₃) in agreement with the title compound.

[0531] Compound 37

[0532] N^(α) (Bzl)(3-Boc-amino propylene) (R)Alanine

[0533] A solution of 5.25 g of N^(α) (Bzl)(2-Boc-amino ethylene)(R)Alanine methyl ester (Compound 35) in MeOH was hydrolyzed by NaOH7.5N (Procedure 7). Yield 100%

[0534] (α)_(D)=−0.5 (C=1, MeOH)

[0535] TLC (Method D) Rf=0.51 (one spot)

[0536] NMR (CDCl₃) in agreement with the title compound.

[0537] Compound 38

[0538] N^(α) (3-Boc-amino propylene) (S)Alanine.HCl

[0539] A solution of 4.47 g of N^(α) (Bzl)(3-Boc-amino propylene)(S)Alanine (Compound 35) in MeOH was hydrogenated on Pd/C (Procedure 8).Yield 75%

[0540] TLC (Method A) Rf=0.42 (one spot)

[0541] NMR (CDCl₃) in agreement with the title compound.

[0542] Compound 39

[0543] N^(α) (6-Boc-amino hexylene) (S)Alanine.HCl

[0544] A solution of 5 g of N^(α) (Bzl)(4-Boc-amino hexylene) (S)Alanine(Compound 36) in MeOH-DMF was hydrogenated on Pd/C (Procedure 8). Yield64.5% of white solid, mp 134-136° C.

[0545] TLC (Method A) Rf=0.39 (one spot)

[0546] NMR (CDCl₃) in agreement with the title compound

[0547] Compound 40

[0548] N^(α) (3-Boc-amino propylene)(R)Alanine.HCl

[0549] A solution of 5 g of N^(α) (Bzl)(4-Boc-amino propylene)(R)Alanine (Compound 37) in MeOH-DMF was hydrogenated on Pd/C (Procedure8). Yield 79.1%

[0550] TLC (Method A) Rf=0.39 (one spot)

[0551] NMR (CDCl₃) in agreement with the title compound.

EXAMPLE 8

[0552] Compound 41

[0553] N^(α) (Fmoc)(3-Boc-amino protylene) (S)Alanine

[0554] A solution of 2.82 g of N^(α) (Bzl)(4-Boc-amino propylene(S)Alanine. HCl (Compound 38) in H₂O-ACN was reacted with FmocOSu(Procedure 9). Yield 75% of white solid, mp 70-72° C.

[0555] TLC (Method D) Rf=0.65 (one spot)

[0556] NMR (CDCl₃) in agreement with the title compound. ElementalAnalysis: % C % H % N Found: 66.40 6.78 5.63 Calc: 66.65 6.88 5.93

EXAMPLE 9

[0557] Compound 42

[0558] N^(α) (Fmoc)(6-Boc-amino hexylene) (S)Alanine

[0559] A solution of 3.25 g of N^(α) (Bzl)(4-Boc-amino hexylene)(S)Alanine .HCl (Compound 39) in H₂O-ACN was reacted with FmocOSu(Procedure 9). Yield 72.8% of white solid, mp 70-72° C.

[0560] TLC (Method D) Rf=0.7 (one spot)

[0561] NMR (CDCl₃) in agreement with the title compound. ElementalAnalysis: % C % H % N Found: 68.37 7.40 5.23 Calc: 68.21 7.50 5.49

[0562] HPLC (Method G) 90%

EXAMPLE 10

[0563] Compound 43

[0564] N^(α) (Fmoc)(3-Boc-amino Dropylene)(R)Alanine

[0565] A solution of 2.82 g of N^(α) (Bzl)(4-Boc-amino propylene)(S)Alanine .HCl (Compound 40) in H₂O-ACN was reacted with FmocOSu(Procedure 9). Yield 75.9% of white solid, mp 70-72° C.

[0566] TLC (Method D) Rf=0.5 (one spot)

[0567] NMR (CDCl₃) in agreement with the title compound. ElementalAnalysis: % C % H % N Found: 66.4 6.78 5.63 Calc: 66.65 6.88 5.93

EXAMPLE 11

[0568] Compound 44

[0569] N-(2-(benzylthio)ethylene)qlycine ethyl ester

[0570] The title compound was prepared according to procedure 13 fromethyl bromo acetate.

[0571] Yield 75% of colorless oil.

[0572] NMR (CDCl₃) in agreement with the title compound.

[0573] Elemental analysis-calculated: C-61.16, H-7.70, N-3.96 found:C-61.45, H-8.03, N-3.49.

[0574] Compound 45

[0575] N-(3-(benzylthio)propylene)glycine methyl ester

[0576] The title compound was prepared according to procedure 13 frommethyl bromo acetate.

[0577] Yield 74% of colorless oil.

[0578] NMR (CDCl₃) in agreement with the title compound.

EXAMPLE 12

[0579] Compound 46

[0580] N-(2-(benzylthio)ethylene)(S)leucine methyl ester

[0581] The title compound was prepared according to procedure 13 fromthe Triflate of (R) leucine methyl ester (Procedure 5).

[0582] Yield 70% of colorless oil.

[0583] NMR (CDCl₃) in agreement with the title compound.

[0584] Elemental analysis-calculated: C-65.05, H-8.53, N-4.74; found:C-66.29, H-9.03, N-4.49.

[0585] (a)_(D14)=−51.2° (C 0.94,DCM).

EXAMPLE 13

[0586] Compound 47

[0587] N-(3-(benzylthio)propylene)(S)leucine methyl ester

[0588] The title compound was prepared according to procedure 13 fromthe Triflate of (R)leucine methyl ester (Procedure 5).

[0589] Yield 60% of colorless oil.

[0590] NMR (CDCl₃) in agreement with the title compound.

[0591] Elemental analysis-calculated: C-65.98, H-8.79, N-4.53; found:C-67.09, H-9.20, N-4.54.

[0592] (a)_(D23)=−17.4° (C 1.44, DCM)

[0593] Compound 48

[0594] N-(2-(benzylthio)ethylene)(S)Phenylalanine methyl ester

[0595] The title compound was prepared according to procedure 13 fromthe Triflate of (R)phenyl lactic acid methyl ester (Procedure 5).

[0596] Yield 82% of white crystals.

[0597] m.p.=48-49° C.

[0598] NMR (CDCl₃) in agreement with the title compound.

[0599] Elemental analysis-calculated: C-69.27, H-7.04, N-4.25 ; found:C-69.S5, H-7.21, N-4.08.

[0600] (a)_(D14)=−23.3° (C=1.01, DCM).

[0601] Compound 49

[0602] N-(3-(benzylthio)propylene)(S)phenylalanine methyl ester

[0603] The title compound was prepared according to procedure 13 fromthe Triflate of (R)phenyl lactic acid methyl ester (Procedure 5).

[0604] Yield 71% of white crystals.

[0605] m.p.=38-39° C.

[0606] NMR (CDCl₃) in agreement with the title compound.

[0607] Elemental analysis-calculated: C-69.94, H-7.34, N-4.08; found:C-69.66, H-7.39,

[0608] N-4.37.

[0609] (a)_(D26)=+2.0° (C 1.00, DCM)

[0610] Compound 50

[0611] N-(4-(benzylthio)butylene)(S)phenylalanine methyl ester

[0612] The title compound was prepared according to procedure 13 fromthe Triflate of (S)phenyl lactic acid methyl ester (Procedure 5).

[0613] Yield 81% of colorless oil.

[0614] NMR (CDCl₃) in agreement with the title compound.

[0615] Elemental analysis-calculated: C-70.55, H-7.61, N-3.92 ; found:C-70.51, H-7.69,

[0616] N-4.22.

[0617] (a)_(D26)=+4.9° (C 1.00, DCM)

EXAMPLE 14

[0618] Compound 51

[0619] Boc-N-(2-(benzylthio)ethylene)glycine

[0620] The title compound was prepared from Compound 44 by hydrolysisaccording to Procedure 11.

[0621] Yield 88% of white crystals.

[0622] m.p.=71-72° C.

[0623] NMR (CDCl₃) in agreement with the title compound.

[0624] Elemental analysis—calculated: c-59.05, H-7.12, N-4.30 ; found:C-59.39, H-7.26, N-4.18.

EXAMPLE 15

[0625] Compound 52

[0626] Boc-N-(2-(benzylthio)ethylene)(S)phenylalanine

[0627] The title compound was prepared from Compound 48 by hydrolysisaccording to Procedure 11.

[0628] Yield 78% of white crystals.

[0629] m.p.=82-83° C.

[0630] NMR (CDCl₃) in agreement with the title compound.

[0631] (a)_(D25)=−105.9° (C 1.01, DCM).

EXAMPLE 16

[0632] Compound 53

[0633] Boc-N-(3-(benzylthio)propylene)(S)phenylalanine

[0634] The title compound was prepared from Compound 49 by hydrolysisaccording to Procedure 11.

[0635] Yield 99% of white crystals.

[0636] m.p.=63-64° C.

[0637] NMR (CDCl₃) in agreement with the title compound.

[0638] (a)_(D25)=−87.4° (C 1.01, DCM)

EXAMPLE 17

[0639] Compound 54

[0640] Boc-L-phenylalanyl-N-(2-(benzylthio)-ethylene)glycine ethyl ester

[0641] Boc-L-Phe was coupled to N-(2-(benzylthio)-ethylene)glycine ethylester (Compound 44) according to Procedure 12.

[0642] Yield 32% of colorless oil.

[0643] NMR (CDCl₃) in agreement with the title compound.

[0644] Elemental analysis—calculated: C-64.77, H-7.25, N-5.60 ; found:C-64.39, H-7.02, N-5.53.

[0645] (a)_(D16)=+4.5° (C 0.88, DCM).

EXAMPLE 18

[0646] Compound 55

[0647] Boc-L-phenylalanyl-N-(2-(benzylthio)-ethylene)(S)phenylalaninemethyl ester

[0648] Boc-7L-Phe was coupled to N-(2-(benzylthio)ethylene)(S)phenylalanine methyl ester (Compound 48) according to Procedure 12.

[0649] Yield 46% of colorless oil.

[0650] NMR (CDCl₃) in agreement with the title compound.

[0651] (a)_(D26)=−115.9° (C 1.0, CHCl₃)

[0652] Compound 56

[0653] N-Bzl-β-alanine t-butyl ester

[0654] A solution of 6.16 g of β-alanine t-butyl ester acetate in 150 mLwater was reacted with benzaldhyde (Procedure 2) to give 4.5 g, 64.5%yield

[0655] TLC (Method A) Rf=0.78 (one spot)

[0656] NMR (CDCl₃) in agreement with the title compound.

[0657] Compound 57

[0658] N-Bzl-γ-amino butyric acid t-butyl ester

[0659] A solution of 6.58 g of γ-aminobutyric acid t-butyl ester acetatein 150 mL water was reacted with benzaldhyde (Procedure 2) to give 4.24g, 57.9% yield

[0660] TLC (Method A) Rf=0.74 (one spot)

[0661] NMR (CDCl₃) in agreement with the title compound.

[0662] Compound 58

[0663] N^(α) (Bzl)(2-t-butyl carboxy ethylene)glycine benzyl ester

[0664] A solution of 3.53 g of N-Bzl-β-alanine t-butyl ester (Compound56) in DMF was reacted with 2.61 mL benzyl bromoacetate (Procedure 17).Yield 86.9%

[0665] TLC (Method F) Rf=0.95 (one spot)

[0666] NMR (CDCl₃) in agreement with the title compound.

[0667] Compound 59

[0668] N^(α) (Bzl)(3-t-butyl carboxy propylene)glycine benzyl ester

[0669] A solution of 3.53 g of N-Bzl-γ-aminobutyric acid t-butyl ester(Compound 57) in DMF was reacted with 2.61 mL benzyl bromoacetate(Procedure 17). Yield 83%

[0670] TLC (Method F) Rf=0.92 (one spot)

[0671] NMR (CDCl₃) in agreement with the title compound.

[0672] Compound 60

[0673] N^(α) (2-t-butyl carboxy ethylene)glycine

[0674] A solution of N^(α) (Bzl)(2-t-butyl carboxy ethylene)glycinebenzyl ester (Compound 58) in MeOH was hydrogenated (Procedure 8). Yield87.8%

[0675] TLC (Method A) Rf=0.56 (one spot)

[0676] NMR (CDCl₃) in agreement with the title compound.

[0677] Compound 61

[0678] N^(α) (3-t-butyl carboxy propylene)glycine

[0679] A solution of N^(α) (Bzl)(3-t-butyl carboxy propylene)glycinebenzyl ester (Compound 59) in MeOH was hydrogenated (Procedure 8). Yield94%

[0680] TLC (Method A) Rf=0.3 (one spot)

[0681] NMR (CDCl₃) in agreement with the title compound.

EXAMPLE 19

[0682] Compound 62

[0683] N^(α)(Fmoc)(2-t-butyl carboxy ethylene)glycine

[0684] A solution of N^(α) (2-t-butyl carboxy ethylene)glycine (Compound60) in H₂O:Et₃N was reacted with FmocOSu (Procedure 9). Yield 90%

[0685] TLC (Method D) Rf=0.5 (one spot)

[0686] NMR (CDCl₃) in agreement with the title compound. ElementalAnalysis: % C % H % N Found: 67.38 6.34 3.11 Calc: 67.75 6.40 3.29

EXAMPLE 20

[0687] Compound 63

[0688] N^(α)(Fmoc)(3-t-butyl carboxy propylene)glycine

[0689] A solution of N^(α) (3-t-butyl carboxy propylene)glycine(Compound 61) in H₂O:Et₃N was reacted with FmocOSu (Procedure 9). Yield82%

[0690] TLC (Method D) Rf=0.58 (one spot)

[0691] NMR (CDCl₃) in agreement with the title compound. ElementalAnalysis: % C % H % N Found: 68.29 6.83 3.88 Calc: 68.32 6.65 3.19

[0692] Compound 64

[0693] (R)-O-Trf-3-Phenyllactic acid benzyl ester

[0694] To a cooled solution of Trf₂O and pyridine in dry DCM (Procedure5), a solution of 5.3 g of (R)-3-Phenyllactic acid benzyl ester wasadded. After the workup (Procedure 5), the yield was 91.43%. The productwas used immediately or kept in a cold desiccator under Ar.

[0695] Compound 65

[0696] N^(α)(Bzl)(2-t-butyl carboxy ethylene)(S) Phenylalanine benzylester

[0697] A solution of 5.48 g of N-Bzl-β-alanine t-butyl ester (Compound56) in DCM was reacted with 7.35 g of (R)-O-Trf-3-Phenyllactic acidbenzyl ester (

[0698] Compound 64) in dry DCM (Procedure 6). After workup the crudeproduct was purified by flash chromatography. PE:EtOAc (4:1) 1.5 L.After solvent evaporation under vacuum, the product was dried undervacuum.

[0699] Yield 71.5%

[0700] TLC (Method C) Rf=0.77 (one spot)

[0701] (α)_(D)=−62.7 (C=1, MeOH)

[0702] NMR (CDCl₃) in agreement with the title compound.

[0703] Compound 66

[0704] N^(α)(2-t-butyl carboxy ethylene)(S) Phenylalanine

[0705] A solution of 6.3 g of N^(α)(Bzl)(2-t-butyl carboxyethylene)(S)Phenylalanine benzyl ester (Compound 65) in MeOH washydrogenated (Procedure 8). Yield 48.6%

[0706] TLC (Method A) Rf=0.52-0.54 (one spot)

[0707] NMR (CDCl₃) in agreement with the title compound.

EXAMPLE 21

[0708] Compound 67

[0709] N^(α)(Fmoc)(2-t-butyl carboxy ethylene)(S)Phenylalanine

[0710] A solution of 2.13 g of N^(α)(2-t-butyl carboxyethylene)(S)Phenylalanine (Compound 66) in H₂O:Et₃N was reacted withFmocOSu (Procedure 9). Yield 38%

[0711] TLC (Method D) Rf=0.77 (one spot)

[0712] NMR (CDCl₃) in agreement with the title compound. ElementalAnalysis: % C % H % N Found: 71.92 639 2.87 Calc: 72.21 6.45 2.72

[0713] HPLC (Method G) 93%

[0714] Compound 68

[0715] N^(α) (Bzl)(2-Poc amino ethylene)glycine benzyl ester

[0716] Elemental analysis—calculated: C-59.05, H-7.12, N-4.30 ; found:C-59.39, H-7.26, N-4.18.

EXAMPLE 15

[0717] Compound 52

[0718] Boc-N-(2-(benzylthio)ethylene)(S)phenylalanine

[0719] The title compound was prepared from Compound 48 by hydrolysisaccording to Procedure 11.

[0720] Yield 78% of white crystals.

[0721] m.p.=82-83° C.

[0722] NMR (CDCl₃) in agreement with the title compound.

[0723] (a)_(D25)=−105.9° (C 1.01, DCM).

EXAMPLE 16

[0724] Compound 53

[0725] Boc-N-(3-(benzylthio)propylene)(S)Phenylalanine

[0726] The title compound was prepared from Compound 49 by hydrolysisaccording to Procedure 11.

[0727] Yield 99% of white crystals.

[0728] m.p.=63-64° C.

[0729] NMR (CDCl₃) in agreement with the title compound.

[0730] (a)_(D25)=−87.4° (C 1.01, DCM).

EXAMPLE 17

[0731] Compound 54

[0732] Boc-L-phenylalanyl-N-(2-(benzylthio)-ethylene)glycine ethyl ester

[0733] Boc-L-Phe was coupled to N-(2-(benzylthio)-ethylene)glycine ethylester (Compound 44) according to Procedure 12.

[0734] Yield 32% of colorless oil.

[0735] NMR (CDCl₃) in agreement with the title compound.

[0736] Elemental analysis-calculated: C-64.77, H-7.25, N-5.60; found:C-64.39, H-7.02, N-5.53.

[0737] (a)_(D16)=+4.5° (C 0.88, DCM).

EXAMPLE 18

[0738] Compound 55

[0739] Boc-L-phenylalanyl-N-(2-(benzylthio)-ethylene)(S)phenylalaninemethyl ester

[0740] NMR (CDCl₃) in agreement with the title compound.

[0741] Compound 73

[0742] N^(α)(3-Boc amino propylene)glycine

[0743] A solution of 0.025 mole of N^(α)(Bzl)(3-Boc aminopropylene)glycine benzyl ester (Compound 69) in 60 mL MeOH washydrogenated (Procedure 8). Yield 74% of white solid. mp 214-6° C.

[0744] TLC (Method A) Rf=0.27 (one spot)

[0745] NMR (CDCl₃) in agreement with the title compound.

[0746] Compound 74

[0747] N^(α)(4-Boc amino butylene)glycine

[0748] A solution of 0.025 mole of N^(α) (Bzl)(4-Boc aminobutylene)glycine benzyl ester (Compound 70) in 60 mL MeOH washydrogenated(Procedure 8). Yield 89.5% of white solid. mp 176-8° C.

[0749] TLC (Method A) Rf=0.23 (one spot)

[0750] NMR (CDCl₃) in agreement with the title compound.

[0751] Compound 75

[0752] N^(α)(6-Boc amino hexylene)glycine

[0753] A solution of 0.025 mole of N^(α) (Bzl)(6-Boc aminohexylene)glycine benzyl ester (Compound 71) in 60 mL MeOH washydrogenated (Procedure 8). Yield 80% of white solid. mp 172-4° C.

[0754] TLC (Method A) Rf=0.26 (one spot)

[0755] NMR (CDCl₃) in agreement with the title compound.

EXAMPLE 22

[0756] Compound 76

[0757] N^(α)(Fmoc)(2-Boc amino ethylene)glycine

[0758] A solution of 0.02 mole of N^(α) (2-Boc amino ethylene)glycine(Compound 72) in H₂O:Et₃N was reacted with FmocOSu (Procedure 9). Yield80% of white solid. mp 130-132° C. TLC (Method D) Rf=0.5 (one spot)

[0759] NMR (CDCl₃) in agreement with the title compound. ElementalAnalysis: % C % H % N Found: 65.18 6.11 5.91 Calc: 65.43 6.40 6.63

EXAMPLE 23

[0760] Compound 77

[0761] N^(α)(Fmoc)(3-Boc amino propylene)glycine

[0762] A solution of 0.02 mole of N^(α)(Fmoc)(3-Boc aminopropylene)glycine (Compound 73) in H₂O:Et₃N was reacted with FmocOSu(Procedure 9). Yield 85% of white solid. mp 125° C.

[0763] TLC (Method D) Rf=0.5-0.6 (one spot)

[0764] NMR (CDCl₃) in agreement with the title compound. ElementalAnalysis: % C % H % N Found: 66.05 6.65 6.00 Calc: 66.06 6.65 6.16

EXAMPLE 24

[0765] Compound 78

[0766] N^(α)(Fmoc)(4-Boc amino butylene)glycine

[0767] A solution of 0.02 mole of N^(α)(Fmoc)(4-Boc aminobutylene)glycine (Compound 74) in H₃O:Et₃N was reacted with FmocOSu(Procedure 9). Yield 79.4% of white solid. mp 150-152° C.

[0768] TLC (Method D) Rf=0.42-0.47 (one spot)

[0769] NMR (CDCl₃) in agreement with the title compound. ElementalAnalysis: % C % H % N Found: 66.35 6.84 5.77 Calc: 66.06 6.88 5.98

EXAMPLE 25

[0770] Compound 79

[0771] N^(α)(Fmoc)(6-Boa amino hexylene)glycine

[0772] A solution of 0.02 mole of N^(α(Fmoc)()6-Boc aminohexylene)glycine (Compound 75) in H₂O:Et₃N was reacted with FmocOSu(Procedure 9). Yield 81.5% of white solid. mp 78-80° C. TLC (Method D)Rf0.7 (one spot)

[0773] NMR (CDCl₃) in agreement with the title compound. ElementalAnalysis: % C % H % N Found: 68.02 7.08 5.37 Calc: 67.72 7.31 5.64

Synthetic Esamples

[0774] Two series of octapeptide somatostatin analogs of the presentinvention were synthesized, characterized, and tested for biologicalactivity.

[0775] 1) The first series of compounds corresponds to the generalFormula (XIVb); this series comprises compounds of the specific formula

H-(D)Phe-R⁶-Phe-(D)Trp-Lys-Thr-R¹¹-Thr-NH₂

[0776] wherein R⁶ and R¹¹ are N^(α) ω-functionalized alkylene amino acidbuilding units.

[0777] 2) The second series of compounds corresponds to the generalFormula (XVIc); this series comprises compounds of the specific formula

H-(D)Phe-R⁶-Phe-(D)Trp-Lys-R¹⁰-Thr-NH₂

[0778] wherein R⁶ and R¹⁰ are N^(α) ω-functionalized alkylene amino acidbuilding units.

[0779] The structures of these novel synthetic peptide analogs intowhich N^(α) ω-functionalized amino acid building units wereincorporated, are summarized in Tables 7 and 8. In both series, thebuilding units used were glycine building units in which the bridginggroups, attached via the alpha nitrogens to the peptide backbone, werevaried.

[0780] For the sake of simplicity, these two series are referred toherein as the SST Gly⁶,Gly¹¹ and SST Gly⁶,Gly¹⁰ series, respectively.

[0781] In each series, the position of the cyclization points wasconstant, while the length and direction of the bridge was varied. Thus,C2,N2 refers to a bridge consisting of an amide bond in which thecarbonyl group is closer to the amino end of the peptide and whichcontains two methylene groups between the bridge amide and each of thebackbone nitrogens involved in the bridge.

[0782] Peptide assembly was carried out either manually or with anautomatic peptide synthesizer (Applied Biosystems Model 433A). Followingpeptide assembly, de-protection of bridging groups that form thecyclization arms was carried out with Pd(PPh₃)₄ (palladium tetrakistriphenyl phosphine) in the case of Allyl/Alloc protecting groups orwith TFA in the case of tBu/Boc protecting groups. For obtaining thelinear (non-cystalized) analog, the peptides were cleaved from the resinat this stage. Cyclization of the peptides was carried out with PyBOP.Cleavage of the peptides from the polymeric support was carried out withsuitable reagents depending on the type of resin used, e.g., with TFAfor Rink amide type resins and with HF for mBHA (para-methyl benzhydrylamine) type resins the crude products were characterized by analyticalHPLC. The peptides were purified by preparative reversed phase HPLC. Thepurified products where characterized by analytical HPLC, massspectroscopy, and amino acid analysis. TABLE 7 SST Gly⁶, Gly¹¹ ExampleBridging Compound Crude No. Groups Number Method Yield 26 C1, N2 CyclicDE-3-32-4 1 NA** 27 C1, N2 Linear DE-3-32-2 1 NA 28 C1, N3 Cyclic PTR3004 2 79 mg 29 C1, N3 Linear PTR 3005 2 34 mg 30 C2, N2 Cyclic PTR 30021 NA 31 C2, N2 Linear PTR 3001 1 NA 32 C2, N3 Cyclic PTR 3007 2 40 mg 33C2, N3 Linear PTR 3008 2 40 mg 34 N2, C2 Cyclic YD-9-166-1 2 NA 35 N2,C2 Linear YD-9-168-1 2 NA 36 N3, C2 Cyclic PTR 3010 2 100 mg 37 N3, C2Linear PTR 3011 2 NA 38 Linear* PTR 3003 3 96 mg

[0783] Table 7 Methods

[0784] 1) Manual synthesis on mBHA resin. HF cleavage.

[0785] 2) Manual synthesis on Rapp tentagel resin. TFA cleavage.

[0786] 3) Rink amide resin; assembly in automated peptide synthesizer,0.1 mmol scale. TABLE 8 SST Gly⁶, Gly¹⁰ Example Bridging Compound CrudeNo. Groups Number Method Yield 39 C1, N2 Cyclic YD-9-171-3 1 20 mg 40C1, N2 Linear YD-9-171-2 1 10 mg 41 C1, N3 Cyclic YD-9-175-3 1 44.9 mg  42 C1, N3 Linear YD-9-175-2 1 25.4 mg   43 C2, N2 Cyclic PTR 3019 1 40mg 44 C2, N2 Linear PTR 3020 1 26 mg 45 C2, N3 Cyclic YD-5-28-3 3 101.5mg   46 C2, N3 Linear YD-5-28-2 3 48.3 mg   47 N2, C2 Cyclic PTR 3016 260 mg 48 N2, C2 Linear PTR 3017 2 40 mg 49 N3, C2 Cyclic YS-8-153-1 2 93mg 50 N3, C2 Linear YS-8-152-1 2 54 mg 51 *Linear PTR 3021 1 100 mg **Acetylated Des-D-Phe⁵ 52 N3, C2 Cyclic PTR 3013 67 mg 53 N3, C2 LinearPTR 3014 48 mg

[0787] Table 8 Methods

[0788] 1) Assembly in automated peptide synthesizer; 0.1 mmol scale.(HBTU).

[0789] 2) Manual synthesis; PyBrop.

[0790] 3) Assembly in automated peptide synthesizer, 0.25 mmol scale.(HBTU).

[0791] Synthesis of SST Gly⁶, Gly¹⁰ N3,C2:

[0792] Five grams of Rink amide resin (NOVA) (0.49 mmol/g), were swelledin N-methylpyrrolidone (NMP) in a reaction vessel equipped with asintered glass bottom and placed on a shaker. The Fmoc protecting groupwas removed from the resin by reaction with 20% piperidine in NMP (2times 10 minutes, 25 ml each). Fmoc removal was monitored by ultravioletabsorption measurement at 290 nm. A coupling cycle was carried out withFmoc-Thr(OtBu)-OH (3 equivalents) PyBrop (3 equivalents) DIEA (6equivalents) in NMP (20 ml) for 2 hours at room temperature. Reactioncompletion was monitored by the qualitative ninhydrin test (Kaisertest). Following coupling, the peptide-resin was washed with NMP (7times with 25 ml NMP, 2 minutes each). Capping was carried out byreaction of the peptide-resin with acetic anhydride (capping mixture:HOBt 400 mg, NMP 20 ml, acetic anhydride 10 ml, DIEA 4.4 ml) for 0.5hours at room temperature. After capping, NMP washes were carried out asabove (7 times, 2 minutes each). Fmoc removal was carried out as above.Fmoc-Phe-OH was coupled in the same manner, and the Fmoc group removed,as above. The peptide resin was reacted with Fmoc-Gly-C2 (Allyl)building unit: coupling conditions were as above. Fmoc removal wascarried out as above. Fmoc-Lys(Boc)-OH was coupled to the peptide resinby reaction with HATU (3 equivalents) and DIEA (6 equivalents) at roomtemperature overnight and then at 50° C. for one hour. Additional DIEAwas added during reaction to maintain a basic medium (as determined bypH paper to be about 9). This coupling was repeated. Coupling completionwas monitored by the Fmoc test (a sample of the peptide resin was takenand weighed, the Fmoc was removed as above, and the ultravioletabsorption was measured). Fmoc-D-Trp-OH was coupled to the peptide resinwith PyBrop, as described above. Following Fmoc removal, Fmoc-Phe-OH wascoupled in the same way. Synthesis was continued with one-fifth of thepeptide resin.

[0793] Following Fmoc removal, the second building unit was introduced:Fmoc-Gly-N3(Alloc)-OH by reaction with PYErop, as described above.Capping was carried out as described above. Following Fmoc removal, thepeptide-resin was divided into two equal portions. Synthesis wascontinued with one of these portions. Boc-D-Phe-OH was coupled byreaction with HATU, as described above for Fmoc-Lys(Boc)-OH. Capping wascarried out as above.

[0794] The Allyl and Alloc protecting groups were removed by reactionwith Pd(PPh₃)₄ and acetic acid 5%, morpholine 2.5% in chloroform, underargon, for 2 hours at room temperature. The peptide resin was washedwith NMP as above. Two-thirds of the resin were taken for cyclization.Cyclization was carried out with PyBOP 3 equivalents, DIEA 6equivalents, in NMP, at room temperature overnight. The peptide resinwas washed and dried. The peptide was cleaved from the resin by reactionwith TFA 81.5%, phenol 5%, water 5%, EDT 2.5%, TIS(tri-isopropyl-silane) 1%, and 5% methylene chloride, at 0° C. for 15minutes and 2 hours at room temperature under argon. The mixture wasfiltered into cold ether (30 ml, 0° C.) and the resin was washed with asmall volume of TFA. The filtrate was placed in a rotary evaporator andall the volatile components were removed. An oily product was obtained.It was triturated with ether and the ether decanted, three times. Awhite powder was obtained. This crude product was dried. The weight ofthe crude product was 93 mg.

Physiological Examples EXAMPLE 54

[0795] Bradykinin Antagonist Assay (Displacement of (³H)dopamine releasefrom PC 12 cells)

[0796] Novel backbone cyclized peptide analogs of the present inventionwere assayed in vitro for bradykinin antagonist activity by protectionof (³H)dopamine release from PC 12 cells that express bradykininreceptors. PC12 cells were grown in Dulbecco Modified Eagle's mediumwith high glucose, supplemented with 10% horse serum, 5% fetal calfserum, 130 units/ml penicillin and 0.1 mg/ml streptomycin. Forexperiments, cells were removed from the medium using 1 mmole EDTA andreplated on collagen coated-12-well plates and assayed 24 hr later.Release of (³H)dopamine was determined as follows: cells were incubatedfor 1.5 hr at 37° C. with 0.5 ml of growth medium and 0.85 ml (³H)DA (41Ci/mmole) and 10 mg/ml pargyline followed by extensive washing withmedium (3×1 ml) and release buffer consisting of (mM): 130 NaCl; 5 KCl;25 NaHCO₃; 1 NaH₂PO₄10 glucose and 1.8 CaCl₂. In a typical experiment,cells were incubated with 0.5 ml buffer for 5 consecutive incubationperiods of 3 min each at 37° C. Spontaneous (³H)DA release was measuredby collecting the medium released by the cells successively for thefirst 3 min period. Antagonists were added to the cells 3 min prior tostimulation (at the second period), and stimulation of (³H)DA release by100 nmole of bradykinin are monitored during the 3 period by 60 mmoleKCl. The remaining of the (³H)DA was extracted from the cells by overnight incubation with 0.5 ml 0.1 N HCl. (³H)DA release during each 3 minperiod was expressed as a % of the total (³H)DA content of the cells.Net evoked release was calculated from (³H)DA release during stimulationperiod after subtracting basal (³H)DA release in the preceding baselineperiod if not indicated otherwise.

[0797] At 10⁻⁶ M, Example 1 showed 30% inhibition of BK activity,Example 4 showed 17% inhibition of BK activity. Note, the noncyclized(control) peptide of example 2 showed 0% inhibition of BK activity.

EXAMPLE 55

[0798] Bradykinin Antagonist Assay (Guinea-Pig Assay)

[0799] The ileum of the guinea-pig was selected as the preparation forthe bioassay. This tissue contains predominantly BK₂ receptors. Thepreparation consists of the longitudinal muscle layer with the adheringmesenteric plexus. The isolated preparation was kept in Krebs solutionand contractions were measured with an isometric force transducer. Theguinea-pig ileum is highly sensitive to BK, with EC₅₀ at 2×10⁻⁸ M. Atleast two control responses to BK (2×10⁻⁸ M) were measured previous tomeasuring the responses of backbone cyclized peptides of the presentinvention. Atropine (1 μm) was always present.

[0800] At 10⁻⁶ M, Example 1 showed 24% inhibition of BK activity,Example 3 showed 10% and Example 4 showed 17% inhibition of BK activity.Note, the noncyclized (control) peptide of example 2 showed 0%inhibition of BK activity.

EXAMPLE 56

[0801] Somatostatin Assay (Receptor Based Screening)

[0802] Initial screening is conducted using ¹²⁵I-labeled SST analogs andpituitary membrane preparations or cell lines. The binding assay isdescribed in Tran, V. T., Beal, M. F. and Martin, J. B. Science,228:294-495, 1985, which is incorporated herein by reference in itsentirety and is optimized with regards to membrane concentration,temperature and time. The assay is sensitive (nM range) and robust.Selectivity will be based on the recent cloning of the five human SSTreceptors. The ability to screen the compounds with regard to bindingand biological activity in mammalian cells should facilitate thedevelopment of subtype-selective analogs. These compounds are useful inthe treatment of specific endocrine disorders and therefore should bedevoid of unwanted side effects.

EXAMPLE 57

[0803] Somatostatin (SST) ASSAY (In vivo Assays)

[0804] The biological effects of SST on growth hormone, insulin andglucagon release is conducted by measuring the levels of these hormonesusing commercially available RIA test kits. Pharmacological effects ofSST in patients with neuroendocrine tumors of the gut will requiredetermination of 5-hydroxyindole acetic acid (for carcinoid) and VIP(for VIPoma). In vivo visualization of SST receptor-positive tumors isperformed as described by Lambert et al., New England J. Med.,323:1246-1249 1990, following i.v. administration of radio-iodinated SSTanalogs.

EXAMPLE 58

[0805] Receptor Binding Specificity of Cyclic Peptide Analogs

[0806] Binding of representative peptides of Examples 39-54 to differentsomatostatin receptors was measured in vitro, in Chinese Hamster Ovary(CHO) cells expressing the various receptors. An example of theselectivity obtained with the cyclic peptides is presented in Table 9.The values presented are percent inhibition of radioactive iodinatedsomatostatin (SRIF-14) binding. TABLE 9 Binding of peptide analogs tosomatostatin receptor subtypes Conc. (M) Somatostatin Receptor (SSTR)Subtype Compound Compound SSTR 2B SSTR 5 Number Description 10⁻⁶ 10⁻⁷10⁻⁸ 10⁻⁶ 10⁻⁷ 10⁻⁸ PTR 3003 Linear 16 3 0 55 20 0 PTR 3004 Cyclic C1,N3 0 0 0 14 0 0 PTR 3005 Linear C1, N3 0 0 0 9 0 0 PTR 3007 Cyclic C2,N3 0 0 0 19 9 0 PTR 3008 Linear C2, N3 0 0 0 15 6 0 PTR 3010 Cyclic N3,C2 0 0 0 63 26 9 PTR 3011 Cyclic N3, C2 0 0 0 27 66 27 Control PeptidesBIM 3503 Pos. Control 81 33 16 92 66 27 PTR 4003 Neg. Control 0 0 0 0 00

EXAMPLE 59

[0807] Resistance to Biodegradation of SST Analogs

[0808] The in vitro biostability of a SST cyclic peptide analog, PTR3002, was measured in human serum, and was compared to the same sequencein a non-cyclic peptide analog (PTR 3001), to octreotide (Sandostatin),and to native somatostatin (SRIF). The results are shown in FIG. 1. Inthis assay, the cyclic peptide in accordance with the present inventionis as stable as octreotide, is more stable than the correspondingnon-cyclic structure, and is much more stable than SRIF. The assay wasbased on HPLC determination of peptide degradation as a function of timeat 37° C.

EXAMPLE 60

[0809] Inhibition of Growth Hormone Release by SST Analogs

[0810] In vivo determination of the pharmacodynamic properties of cyclicpeptide analogs was carried out. Inhibition of Growth Hormone (GH)release as a result of peptide administration was measured. Measurementswere carried out in Sprague-Dawley male rats: peptide analog activitywas compared in this study to SRIF or to octreotide (Sandostatin). Eachgroup consisted of 4 rats. Time course profiles for GH release underconstant experimental conditions were measured.

[0811] Methods

[0812] Adult male Sprague-Dawley rats, specific pathogen free (SPF),weighing 200-350 g, were maintained on a constant light-dark cycle(light from 8:00 to 20:00 h), temperature (21±3° C.), and relativehumidity (55±10%). Laboratory chow and tap water were available adlibitum. On the day of the experiment, rats were anesthetized withpentobarbitone (50 mg/kg). Rats anesthetized with pentobarbitone exhibitlow somatostatin levels in portal blood vessels. (Plotsky, P. M.,Science, 230, 461-463, 1985). A single blood sample (0.6 ml) was takenfrom the exposed cannulated jugular vein for the determination of thebasal GH levels (−15 min). Immediately thereafter the appropriatepeptide pretreatment was administered. The animals received 10 μg/kg ofeither native somatostatin (SRIF) or the synthetic analog octreotide(Sandostatin), or the cyclic peptide analog. A saline solution (0.9%NaCl) was administered as a control. All peptides were administeredsubcutaneously in a final volume of 0.2 ml. Further sampling was carriedout at 15, 30, 60, and 90 minutes after peptide administration.Immediately after the collection of each blood sample, an appropriatevolume (0.6 ml) of saline was administered intravenously. Blood sampleswere collected into tubes containing heparin (15 unites per ml of blood)and centrifuged immediately. Plasma was separated and kept frozen at−20° C. until assayed.

[0813] Rat growth hormone (rGH) [¹²⁵I] levels were determined byappropriate radioimmunoassay kit (Amersham). The standard in this kithas been calibrated against a reference standard preparation (NIH-RP2)obtained from the National Institute of Diabetes and Digestive andKidney Diseases. All samples were measured in duplicate.

EXAMPLE 61

[0814] Lack of Toxicity of Cyclized Peptide Analogs

[0815] PTR 3007 at a dose of 1.5 mg/kg was well tolerated after singleintraperitoneal application. PTR 3013 was not toxic to the rats evenwith doses of 4 mg/kg. These two doses are several orders of magnitudehigher than those needed to elicit the desired endocrine effect. Thepeptides dissolved in saline produced no untoward side effects on thecentral nervous system, cardiovascular system, body temperature, nor onthe periphery of the animals. Rats were observed for 4 hours postadministration of the peptides. PTR 3007 and 3013 produced norespiratory disturbances, did not result in the appearance ofstereotyped behavior, or produce any changes in muscle tone. After 3hours, postmortem examination did not detect any abnormality in theliver, kidneys, arteries and veins, gastrointestinal tract, lungs,genital system, nor the spleen.

What is claimed is:
 1. A backbone cyclized peptide analog comprising apeptide sequence that incorporates at least two building units, each ofwhich contains one nitrogen atom of the peptide backbone connected to abridging group comprising a disulfide, amide, thioether, thioester,imine, ether, or alkene bridge, wherein at least two of said buildingunits are joined together to form a cyclic structure.
 2. The backbonecyclized peptide analog of claim 1 that incorporates at least fourbuilding units, each of which contains one nitrogen atom of the peptidebackbone connected to the bridging groups, wherein at least two pairs ofsaid building units are joined together to form at least two cyclicstructures within said peptide sequence.
 3. The backbone cyclizedpeptide analog of claim 1 wherein at least one of said building units isnot located at the end of the peptide sequence.
 4. The backbone cyclizedpeptide analog of claim 1 wherein none of said building units is locatedat the end of the peptide sequence.
 5. A method for the preparation ofcyclic peptides of the general Formula (I):

wherein: a and b each independently designates an integer from 1 to 8 orzero; d, e, and f each independently designates an integer from 1 to 10;(AA) designates an amino acid residue wherein the amino acid residues ineach chain may be the same or different; E represents a hydroxyl group,a carboxyl protecting group or an amino group, or CO—E can be reduced toCH₂—OH; R and R′ each designates an amino acid side-chain optionallybound with a specific protecting group; and the lines designate abridging group of the Formula: -X-M-Y-W-Z-  (i) or -X-M-Z-  (ii)wherein: one line may be absent; M and W are independently selected fromthe group consisting of disulfide, amide, thioether, thioester, imine,ether, and alkene; and X, Y and Z are each independently selected fromthe group consisting of alkylene, substituted alkylene, arylene, homo-or hetero-cycloalkylene and substituted cycloalkylene; comprising thesteps of incorporating at least one N^(α)-ω-functionalized derivativesof amino acids of Formula (VI):

wherein X is a spacer group selected from the group consisting ofalkylene, substituted alkylene, arylene, cycloalkylene and substitutedcycloalkylene; R′ is an amino acid side chain, optionally bound with aspecific protecting group; B is a protecting group selected from thegroup consisting of alkyloxy, substituted alkyloxy, or aryl carbonyls;and G is a functional group selected from the group consisting ofamines, thiols, alcohols, carboxylic acids and esters, aldehydes,alcohols and alkyl halides; and A is a specific protecting group of G;into a peptide sequence and subsequently selectively cyclizing thefunctional group with one of the side chains of the amino acids in saidpeptide sequence or with another ω-functionalized amino acid derivative.6. The method of claim 5 wherein both lines in Formula (I) are presentand at least four N^(α) ω-functionalized amino acid derivatives areincorporated into the peptide sequence, resulting in a bicycliccompound.
 7. The method of claim 5 wherein E is covalently bound to aninsoluble polymeric support.
 8. The method of claim 5 wherein G is anamine, thiol or carboxyl group.
 9. The method of claim 5 wherein R isselected from the group consisting of CH₃—, (CH₃)₂CH—, (CH₃)₂CHCH₂—,CH₃CH₂CH(CH₃)—, CH₃S(CH₂)₂—, HOCH₂—, CH₃CH(OH)—, HSCH₂—, NH₂C(═O)CH₂—,NH₂C(═O)(CH₂)₂—, NH₂(CH₂)₃—, HOC(═O)CH₂—, HOC(═O)(CH₂)₂—, NH₂(CH₂)₄—,C(NH₂)₂ NH(CH₂)₃—, HO-phenyl-CH₂—, benzyl, methylindole, andmethylimidazole.
 10. The method of claim 5 wherein R′ is selected fromthe group consisting of CH₃—, (CH₃)₂CH—, (CH₃)₂CHCH₂—, CH₃CH₂CH(CH₃)—,CH₃S(CH₂)₂—, HOCH₂—, CH₃CH(OH)—, HSCH₂—, NH₂C(═O)CH₂—, NH₂C(═O)(CH₂)₂—,NH₂(CH₂)₃—, HOC(═O)CH₂—, HOC(═O)(CH₂)₂—, NH₂(CH₂)₄—, C(NH₂)₂NH(CH₂)₃—,HO-phenyl-CH₂—, benzyl, methylindole, and methylimidazole.
 11. A methodfor the preparation of cyclic peptides of the general Formula (II):

wherein d, e and f each independently designates an integer from 1 to10; (AA) designates an amino acid residue wherein the amino acidresidues in each chain may be the same or different; E represents ahydroxyl group, a carboxyl protecting or an amino group, or CO—E can bereduced to CH₂—OH; R is an amino acid side chain optionally bound with aspecific protecting group; and the line designates a bridging group ofthe Formula: -X-M-Y-W-Z-  (i) or -X-M-Z-  (ii) wherein M and W areindependently selected from the group consisting of disulfide, amide,thioether, thioester, imine, ether, and alkene; X, Y and Z are eachindependently selected from the group consisting of alkylene,substituted alkylene, arylene, homo- or hetero-cycloalkylene andsubstituted cycloalkylene; comprising the steps of: incorporating atleast one ω-functionalized amino acid derivative of the general Formula(VI):

wherein X is a spacer group selected from the group consisting ofalkylene, substituted alkylene, arylene, cycloalkylene and substitutedcycloalkylene; R is the side chain of an amino acid; B is a protectinggroup selected from the group consisting of alkyloxy, substitutedalkyloxy, or aryloxy; and G is a functional group selected from thegroup consisting of amines, thiols, alcohols, carboxylic acids andesters or alkyl halides and A is a protecting group thereof; into apeptide sequence and subsequently selectively cyclizing the functionalgroup with one of the side chains of the amino acids in said peptidesequence.
 12. The method of claim 10 wherein G is a carboxyl group or athiol group.
 13. The method of claim 10 wherein R is CH₃—, (CH₃)₂CH—,(CH₃)₂CHCH₂—, CH₃CH₂CH(CH₃)—, CH₃S(CH₂)₂—, HOCH₂—, CH₃CH(OH)—, HSCH₂—,NH₂C(═O)CH₂—, NH₂C(═O)(CH₂)₂—, NH₂(CH₂)₃—, HOC(═O)CH₂—, HOC(═O)(CH₂)₂—,NH₂ (CH₂) ₄—, C(NH₂)₂NH(CH₂)₃—, phenyl-CH₂—, benzyl, methylindole, ormethylimidazole.
 14. The method of claim 10 wherein the peptide iscovalently coupled to an insoluble polymeric support.